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Sample records for alternative differentiation reveals

  1. Gene expression kinetics in individual plasmodial cells reveal alternative programs of differential regulation during commitment and differentiation.

    PubMed

    Rätzel, Viktoria; Marwan, Wolfgang

    2015-05-26

    During its life cycle, the amoebozoon Physarum polycephalum forms multinucleate plasmodial cells that can grow to macroscopic size while maintaining a naturally synchronous population of nuclei. Sporulation-competent plasmodia were stimulated through photoactivation of the phytochrome photoreceptor and the expression of sporulation marker genes was analyzed quantitatively by repeatedly taking samples of the same plasmodial cell at successive time points after the stimulus pulse. Principal component analysis of the gene expression data revealed that plasmodial cells take different trajectories leading to cell fate decision and differentiation and suggested that averaging over individual cells is inappropriate. Queries for genes with pairwise correlated expression kinetics revealed qualitatively different patterns of co-regulation, indicating that alternative programs of differential regulation are operational in individual plasmodial cells. At the single cell level, the response to stimulation of a non-sporulating mutant was qualitatively different as compared to the wild type with respect to the differentially regulated genes and their patterns of co-regulation. The observation of individual differences during commitment and differentiation supports the concept of a Waddington-type quasipotential landscape for the regulatory control of cell differentiation. Comparison of wild type and sporulation mutant data further supports the idea that mutations may impact the topology of this landscape.

  2. Correcting for differential transcript coverage reveals a strong relationship between alternative splicing and organism complexity.

    PubMed

    Chen, Lu; Bush, Stephen J; Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Urrutia, Araxi O

    2014-06-01

    What at the genomic level underlies organism complexity? Although several genomic features have been associated with organism complexity, in the case of alternative splicing, which has long been proposed to explain the variation in complexity, no such link has been established. Here, we analyzed over 39 million expressed sequence tags available for 47 eukaryotic species with fully sequenced genomes to obtain a comparable index of alternative splicing estimates, which corrects for the distorting effect of a variable number of transcripts per species--an important obstacle for comparative studies of alternative splicing. We find that alternative splicing has steadily increased over the last 1,400 My of eukaryotic evolution and is strongly associated with organism complexity, assayed as the number of cell types. Importantly, this association is not explained as a by-product of covariance between alternative splicing with other variables previously linked to complexity including gene content, protein length, proteome disorder, and protein interactivity. In addition, we found no evidence to suggest that the relationship of alternative splicing to cell type number is explained by drift due to reduced N(e) in more complex species. Taken together, our results firmly establish alternative splicing as a significant predictor of organism complexity and are, in principle, consistent with an important role of transcript diversification through alternative splicing as a means of determining a genome's functional information capacity.

  3. Molecular Characterization of the α-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants

    PubMed Central

    Lind, Ulrika; Alm Rosenblad, Magnus; Wrange, Anna-Lisa; Sundell, Kristina S.; Jonsson, Per R.; André, Carl; Havenhand, Jonathan; Blomberg, Anders

    2013-01-01

    The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na+/K+ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions. PMID:24130836

  4. Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

    USGS Publications Warehouse

    Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

    2009-01-01

    The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-?? gene (rtIFN-??2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in na??ve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-??1 and rtIFN-??2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-??2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection. ?? 2008 Elsevier Ltd.

  5. Alternative splicing modulates stem cell differentiation.

    PubMed

    Fu, Ru-Huei; Liu, Shih-Ping; Ou, Chen-Wei; Yu, Hsiu-Hui; Li, Kuo-Wei; Tsai, Chang-Hai; Shyu, Woei-Cherng; Lin, Shinn-Zong

    2009-01-01

    Stem cells have the surprising potential to develop into many different cell types. Therefore, major research efforts have focused on transplantation of stem cells and/or derived progenitors for restoring depleted diseased cells in degenerative disorders. Understanding the molecular controls, including alternative splicing, that arise during lineage differentiation of stem cells is crucial for developing stem cell therapeutic approaches in regeneration medicine. Alternative splicing to allow a single gene to encode multiple transcripts with different protein coding sequences and RNA regulatory elements increases genomic complexities. Utilizing differences in alternative splicing as a molecular marker may be more sensitive than simply gene expression in various degrees of stem cell differentiation. Moreover, alternative splicing maybe provide a new concept to acquire induced pluripotent stem cells or promote cell-cell transdifferentiation for restorative therapies and basic medicine researches. In this review, we highlight the recent advances of alternative splicing regulation in stem cells and their progenitors. It will hopefully provide much needed knowledge into realizing stem cell biology and related applications.

  6. Balance Trees Reveal Microbial Niche Differentiation.

    PubMed

    Morton, James T; Sanders, Jon; Quinn, Robert A; McDonald, Daniel; Gonzalez, Antonio; Vázquez-Baeza, Yoshiki; Navas-Molina, Jose A; Song, Se Jin; Metcalf, Jessica L; Hyde, Embriette R; Lladser, Manuel; Dorrestein, Pieter C; Knight, Rob

    2017-01-01

    Advances in sequencing technologies have enabled novel insights into microbial niche differentiation, from analyzing environmental samples to understanding human diseases and informing dietary studies. However, identifying the microbial taxa that differentiate these samples can be challenging. These issues stem from the compositional nature of 16S rRNA gene data (or, more generally, taxon or functional gene data); the changes in the relative abundance of one taxon influence the apparent abundances of the others. Here we acknowledge that inferring properties of individual bacteria is a difficult problem and instead introduce the concept of balances to infer meaningful properties of subcommunities, rather than properties of individual species. We show that balances can yield insights about niche differentiation across multiple microbial environments, including soil environments and lung sputum. These techniques have the potential to reshape how we carry out future ecological analyses aimed at revealing differences in relative taxonomic abundances across different samples. IMPORTANCE By explicitly accounting for the compositional nature of 16S rRNA gene data through the concept of balances, balance trees yield novel biological insights into niche differentiation. The software to perform this analysis is available under an open-source license and can be obtained at https://github.com/biocore/gneiss. Author Video: An author video summary of this article is available.

  7. Balance Trees Reveal Microbial Niche Differentiation

    PubMed Central

    Morton, James T.; Sanders, Jon; Quinn, Robert A.; McDonald, Daniel; Gonzalez, Antonio; Vázquez-Baeza, Yoshiki; Navas-Molina, Jose A.; Metcalf, Jessica L.; Hyde, Embriette R.; Lladser, Manuel; Dorrestein, Pieter C.

    2017-01-01

    ABSTRACT Advances in sequencing technologies have enabled novel insights into microbial niche differentiation, from analyzing environmental samples to understanding human diseases and informing dietary studies. However, identifying the microbial taxa that differentiate these samples can be challenging. These issues stem from the compositional nature of 16S rRNA gene data (or, more generally, taxon or functional gene data); the changes in the relative abundance of one taxon influence the apparent abundances of the others. Here we acknowledge that inferring properties of individual bacteria is a difficult problem and instead introduce the concept of balances to infer meaningful properties of subcommunities, rather than properties of individual species. We show that balances can yield insights about niche differentiation across multiple microbial environments, including soil environments and lung sputum. These techniques have the potential to reshape how we carry out future ecological analyses aimed at revealing differences in relative taxonomic abundances across different samples. IMPORTANCE By explicitly accounting for the compositional nature of 16S rRNA gene data through the concept of balances, balance trees yield novel biological insights into niche differentiation. The software to perform this analysis is available under an open-source license and can be obtained at https://github.com/biocore/gneiss. Author Video: An author video summary of this article is available. PMID:28144630

  8. Implications for practice: Resurgence and differential reinforcement of alternative responding.

    PubMed

    Bloom, Sarah E; Lambert, Joseph M

    2015-12-01

    During the maintenance stages of differential reinforcement of alternative responding (DRA), failure to reinforce alternative responses could result in a resurgence of problem behavior. However, translational work done with arbitrary human responses suggests that teaching individuals to emit multiple alternative responses in sequential order may facilitate the resurgence of appropriate, rather than problem, behavior. This paper discusses the practical implications of serial DRA training on problem and appropriate behavior resurgence, as presented in the preceding article, "Serial Alternative Response Training As Intervention for Target Response Resurgence." Clinical scenarios as well as implications for self-advocacy and acceptability of behavioral interventions are considered.

  9. Impacts of Alternative Splicing Events on the Differentiation of Adipocytes

    PubMed Central

    Lin, Jung-Chun

    2015-01-01

    Alternative splicing was found to be a common phenomenon after the advent of whole transcriptome analyses or next generation sequencing. Over 90% of human genes were demonstrated to undergo at least one alternative splicing event. Alternative splicing is an effective mechanism to spatiotemporally expand protein diversity, which influences the cell fate and tissue development. The first focus of this review is to highlight recent studies, which demonstrated effects of alternative splicing on the differentiation of adipocytes. Moreover, use of evolving high-throughput approaches, such as transcriptome analyses (RNA sequencing), to profile adipogenic transcriptomes, is also addressed. PMID:26389882

  10. Revealing Numerical Solutions of a Differential Equation

    ERIC Educational Resources Information Center

    Glaister, P.

    2006-01-01

    In this article, the author considers a student exercise that involves determining the exact and numerical solutions of a particular differential equation. He shows how a typical student solution is at variance with a numerical solution, suggesting that the numerical solution is incorrect. However, further investigation shows that this numerical…

  11. Alternative Splicing of G9a Regulates Neuronal Differentiation.

    PubMed

    Fiszbein, Ana; Giono, Luciana E; Quaglino, Ana; Berardino, Bruno G; Sigaut, Lorena; von Bilderling, Catalina; Schor, Ignacio E; Steinberg, Juliana H Enriqué; Rossi, Mario; Pietrasanta, Lía I; Caramelo, Julio J; Srebrow, Anabella; Kornblihtt, Alberto R

    2016-03-29

    Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

  12. When Unified Teacher Pay Scales Meet Differential Alternative Returns

    ERIC Educational Resources Information Center

    Walsh, Patrick

    2014-01-01

    This paper quantifies the extent to which unified teacher pay scales and differential alternatives produce opportunity costs that are asymmetric in math and verbal skills. Data from the Baccalaureate and Beyond 1997 and 2003 follow-ups are used to estimate a fully parametric, selection-corrected wage equation for nonteachers, which is then used to…

  13. An Investigation of Differential Reinforcement of Alternative Behavior without Extinction

    ERIC Educational Resources Information Center

    Athens, Elizabeth S.; Vollmer, Timothy R.

    2010-01-01

    We manipulated relative reinforcement for problem behavior and appropriate behavior using differential reinforcement of alternative behavior (DRA) without an extinction component. Seven children with developmental disabilities participated. We manipulated duration (Experiment 1), quality (Experiment 2), delay (Experiment 3), or a combination of…

  14. An Alternative Method of Thinning Reinforcer Delivery during Differential Reinforcement

    ERIC Educational Resources Information Center

    Roane, Henry S.; Fisher, Wayne W.; Sgro, Gina M.; Falcomata, Terry S.; Pabico, Robert R.

    2004-01-01

    Differential reinforcement of alternative behavior (DRA) may result in rates of reinforcement that are impractical for caregivers to implement; therefore, recent research has examined methods for thinning reinforcer delivery during DRA. In this study, reinforcer delivery was thinned during DRA by restricting access to the participant's alternative…

  15. Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation

    PubMed Central

    Schor, Ignacio E; Fiszbein, Ana; Petrillo, Ezequiel; Kornblihtt, Alberto R

    2013-01-01

    Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts. PMID:23892457

  16. Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation.

    PubMed

    Schor, Ignacio E; Fiszbein, Ana; Petrillo, Ezequiel; Kornblihtt, Alberto R

    2013-08-14

    Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.

  17. Differential Impacts of Alternative Splicing Networks on Apoptosis

    PubMed Central

    Lin, Jung-Chun; Tsao, Mei-Fen; Lin, Ying-Ju

    2016-01-01

    Apoptosis functions as a common mechanism to eliminate unnecessary or damaged cells during cell renewal and tissue development in multicellular organisms. More than 200 proteins constitute complex networks involved in apoptotic regulation. Imbalanced expressions of apoptosis-related factors frequently lead to malignant diseases. The biological functions of several apoptotic factors are manipulated through alternative splicing mechanisms which expand gene diversity by generating discrete variants from one messenger RNA precursor. It is widely observed that alternatively-spliced variants encoded from apoptosis-related genes exhibit differential effects on apoptotic regulation. Alternative splicing events are meticulously regulated by the interplay between trans-splicing factors and cis-responsive elements surrounding the regulated exons. The major focus of this review is to highlight recent studies that illustrate the influences of alternative splicing networks on apoptotic regulation which participates in diverse cellular processes and diseases. PMID:27983653

  18. Rbfox3-regulated alternative splicing of Numb promotes neuronal differentiation during development

    PubMed Central

    Kim, Kee K.; Nam, Joseph

    2013-01-01

    Alternative premRNA splicing is a major mechanism to generate diversity of gene products. However, the biological roles of alternative splicing during development remain elusive. Here, we focus on a neuron-specific RNA-binding protein, Rbfox3, recently identified as the antigen of the widely used anti-NeuN antibody. siRNA-mediated loss-of-function studies using the developing chicken spinal cord revealed that Rbfox3 is required to promote neuronal differentiation of postmitotic neurons. Numb premRNA encoding a signaling adaptor protein was found to be a target of Rbfox3 action, and Rbfox3 repressed the inclusion of an alternative exon via binding to the conserved UGCAUG element in the upstream intron. Depleting a specific Numb splice isoform reproduced similar neuronal differentiation defects. Forced expression of the relevant Numb splice isoform was sufficient to rescue, in an isoform-specific manner, postmitotic neurons from defects in differentiation caused by Rbfox3 depletion. Thus, Rbfox3-dependent Numb alternative splicing plays an important role in the progression of neuronal differentiation during vertebrate development. PMID:23420872

  19. AN INVESTIGATION OF DIFFERENTIAL REINFORCEMENT OF ALTERNATIVE BEHAVIOR WITHOUT EXTINCTION

    PubMed Central

    Athens, Elizabeth S; Vollmer, Timothy R

    2010-01-01

    We manipulated relative reinforcement for problem behavior and appropriate behavior using differential reinforcement of alternative behavior (DRA) without an extinction component. Seven children with developmental disabilities participated. We manipulated duration (Experiment 1), quality (Experiment 2), delay (Experiment 3), or a combination of each (Experiment 4), such that reinforcement favored appropriate behavior rather than problem behavior even though problem behavior still produced reinforcement. Results of Experiments 1 to 3 showed that behavior was often sensitive to manipulations of duration, quality, and delay in isolation, but the largest and most consistent behavior change was observed when several dimensions of reinforcement were combined to favor appropriate behavior (Experiment 4). Results suggest strategies for reducing problem behavior and increasing appropriate behavior without extinction. PMID:21541145

  20. An investigation of differential reinforcement of alternative behavior without extinction.

    PubMed

    Athens, Elizabeth S; Vollmer, Timothy R

    2010-01-01

    We manipulated relative reinforcement for problem behavior and appropriate behavior using differential reinforcement of alternative behavior (DRA) without an extinction component. Seven children with developmental disabilities participated. We manipulated duration (Experiment 1), quality (Experiment 2), delay (Experiment 3), or a combination of each (Experiment 4), such that reinforcement favored appropriate behavior rather than problem behavior even though problem behavior still produced reinforcement. Results of Experiments 1 to 3 showed that behavior was often sensitive to manipulations of duration, quality, and delay in isolation, but the largest and most consistent behavior change was observed when several dimensions of reinforcement were combined to favor appropriate behavior (Experiment 4). Results suggest strategies for reducing problem behavior and increasing appropriate behavior without extinction.

  1. Validation and Interrogation of Differentially Expressed and Alternatively Spliced Genes in African-American Prostate Cancer

    DTIC Science & Technology

    2015-10-01

    RNA and annotated. In addition, we have developed SSOs to manipulate PIK3CD alternative splicing, to correct aberrant splicing leading to production...molecular mechanisms, differential gene expression, alternative RNA splicing, epigenetic alterations, clinical tumor aggressiveness 16. SECURITY...words): Prostate cancer, health disparities among racial groups, molecular mechanisms, differential gene expression, alternative RNA splicing

  2. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  3. Small molecules reveal an alternative mechanism of Bax activation

    PubMed Central

    Brahmbhatt, Hetal; Uehling, David; Al-awar, Rima; Leber, Brian; Andrews, David

    2016-01-01

    The pro-apoptotic protein Bax commits a cell to death by permeabilizing the mitochondrial outer membrane (MOM). To obtain small-molecule probes for elucidating the molecular mechanism(s) of Bax activation, we screened for compounds that induced Bax-mediated liposome permeabilization. We identified five structurally different small molecules that promoted both Bax targeting to and oligomerization at membranes. All five compounds initiated Bax oligomerization in the absence of membranes by a mechanism unlike Bax activation by Bcl-2 homology 3 domain (BH3) proteins. Some of the compounds induced Bax/Bak-dependent apoptosis in cells. Activation of Bax by the most active compound was poorly inhibited by the anti-apoptotic protein Bcl-XL and requires a cysteine residue at position 126 of Bax that is not required for activation by BH3 proteins. Our results reveal a novel pathway for Bax activation independent of pro-apoptotic BH3 proteins that may have important implications for the regulation of Bax activity in cells. PMID:26916338

  4. Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature.

    PubMed

    Jiang, Jianfu; Liu, Xinna; Liu, Chonghuai; Liu, Guotian; Li, Shaohua; Wang, Lijun

    2017-02-01

    Heat stress is one of the primary abiotic stresses that limit crop production. Grape (Vitis vinifera) is a cultivated fruit with high economic value throughout the world, with its growth and development often influenced by high temperature. Alternative splicing (AS) is a widespread phenomenon increasing transcriptome and proteome diversity. We conducted high-temperature treatments (35°C, 40°C, and 45°C) on grapevines and assessed transcriptomic (especially AS) and proteomic changes in leaves. We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping, and alternative donor/acceptor sites were markedly induced under different high temperatures. Among all differential AS events, IR was the most abundant up- and down-regulated event. Moreover, the occurrence frequency of IR events at 40°C and 45°C was far higher than at 35°C. These results indicated that AS, especially IR, is an important posttranscriptional regulatory event during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA-binding proteins SR45, SR30, and SR34 and the nuclear ribonucleic protein U1A gradually rose as ambient temperature increased, which revealed a reason why AS events occurred more frequently under high temperature. After integrating transcriptomic and proteomic data, we found that heat shock proteins and some important transcription factors such as MULTIPROTEIN BRIDGING FACTOR1c and HEAT SHOCK TRANSCRIPTION FACTOR A2 were involved mainly in heat tolerance in grape through up-regulating transcriptional (especially modulated by AS) and translational levels. To our knowledge, these results provide the first evidence for grape leaf responses to high temperature at simultaneous transcriptional, posttranscriptional, and translational levels.

  5. Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature1[OPEN

    PubMed Central

    Jiang, Jianfu; Liu, Xinna; Liu, Guotian; Li, Shaohua

    2017-01-01

    Heat stress is one of the primary abiotic stresses that limit crop production. Grape (Vitis vinifera) is a cultivated fruit with high economic value throughout the world, with its growth and development often influenced by high temperature. Alternative splicing (AS) is a widespread phenomenon increasing transcriptome and proteome diversity. We conducted high-temperature treatments (35°C, 40°C, and 45°C) on grapevines and assessed transcriptomic (especially AS) and proteomic changes in leaves. We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping, and alternative donor/acceptor sites were markedly induced under different high temperatures. Among all differential AS events, IR was the most abundant up- and down-regulated event. Moreover, the occurrence frequency of IR events at 40°C and 45°C was far higher than at 35°C. These results indicated that AS, especially IR, is an important posttranscriptional regulatory event during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA-binding proteins SR45, SR30, and SR34 and the nuclear ribonucleic protein U1A gradually rose as ambient temperature increased, which revealed a reason why AS events occurred more frequently under high temperature. After integrating transcriptomic and proteomic data, we found that heat shock proteins and some important transcription factors such as MULTIPROTEIN BRIDGING FACTOR1c and HEAT SHOCK TRANSCRIPTION FACTOR A2 were involved mainly in heat tolerance in grape through up-regulating transcriptional (especially modulated by AS) and translational levels. To our knowledge, these results provide the first evidence for grape leaf responses to high temperature at simultaneous transcriptional, posttranscriptional, and translational levels. PMID:28049741

  6. Alternative response training, differential reinforcement of other behavior, and extinction in squirrel monkeys (Saimiri sciureus)1

    PubMed Central

    Mulick, J. A.; Leitenberg, H.; Rawson, R. A.

    1976-01-01

    In Experiment I, (a) extinction, (b) extinction plus reinforcement of a discrete alternative response, and (c) differential reinforcement of other behavior were each correlated with a different stimulus in a three-component multiple schedule. The alternative-response procedure more rapidly and completely suppressed behavior than did differential reinforcement of other behavior. Differential reinforcement of other behavior was slightly more effective than extinction alone. In Experiment II, reinforcement of specific alternative behavior during extinction and differential reinforcement of other behavior were used in two components, while one component continued to provide reinforcement for the original response. Once again, the alternative-response procedure was most effective in reducing responding as long as it remained in effect. However, the responding partially recovered when reinforcement for competing behavior was discontinued. In general, responding was less readily reduced by differential reinforcement of other behavior than by the specific alternative-response procedure. PMID:16811914

  7. Differential Reinforcement of Alternative Behavior in Center-Based Classrooms: Evaluation of Pre-Teaching the Alternative Behavior

    ERIC Educational Resources Information Center

    LeGray, Matthew W.; Dufrene, Brad A.; Mercer, Sterett; Olmi, D. Joe; Sterling, Heather

    2013-01-01

    This study investigated the effectiveness of a differential reinforcement of alternative behavior procedure in decreasing disruptive behavior while simultaneously increasing the appropriate behavior of four children of typical development between the ages of 4 and 6 in center-based classrooms. We began with brief functional analyses for each…

  8. On the Lower Alternating Integral of Pontryagin in Linear Differential Games of Pursuit

    NASA Astrophysics Data System (ADS)

    Nikol'skiĭ, M. S.

    1987-02-01

    In this article the concept of the lower alternating integral of Pontryagin, which differs from the analogous concept of A. Azamov (MR 83k: 90142), is introduced. The properties of this object are studied in comparison with those of the alternating integral of Pontryagin. A procedure is given for application of the lower alternating integral of Pontryagin to linear differential games of pursuit, with special consideration for the question of using the lower alternating integral of Pontryagin when there are countably many measurements of the phase vector. The results are illustrated in two known differential games: "the boy and the crocodile" and the "control example of Pontryagin."Bibliography: 18 titles.

  9. Transcriptome analyses of primitively eusocial wasps reveal novel insights into the evolution of sociality and the origin of alternative phenotypes

    PubMed Central

    2013-01-01

    Background Understanding how alternative phenotypes arise from the same genome is a major challenge in modern biology. Eusociality in insects requires the evolution of two alternative phenotypes - workers, who sacrifice personal reproduction, and queens, who realize that reproduction. Extensive work on honeybees and ants has revealed the molecular basis of derived queen and worker phenotypes in highly eusocial lineages, but we lack equivalent deep-level analyses of wasps and of primitively eusocial species, the latter of which can reveal how phenotypic decoupling first occurs in the early stages of eusocial evolution. Results We sequenced 20 Gbp of transcriptomes derived from brains of different behavioral castes of the primitively eusocial tropical paper wasp Polistes canadensis. Surprisingly, 75% of the 2,442 genes differentially expressed between phenotypes were novel, having no significant homology with described sequences. Moreover, 90% of these novel genes were significantly upregulated in workers relative to queens. Differential expression of novel genes in the early stages of sociality may be important in facilitating the evolution of worker behavioral complexity in eusocial evolution. We also found surprisingly low correlation in the identity and direction of expression of differentially expressed genes across similar phenotypes in different social lineages, supporting the idea that social evolution in different lineages requires substantial de novo rewiring of molecular pathways. Conclusions These genomic resources for aculeate wasps and first transcriptome-wide insights into the origin of castes bring us closer to a more general understanding of eusocial evolution and how phenotypic diversity arises from the same genome. PMID:23442883

  10. MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-Seq data

    PubMed Central

    Shen, Shihao; Park, Juw Won; Huang, Jian; Dittmar, Kimberly A.; Lu, Zhi-xiang; Zhou, Qing; Carstens, Russ P.; Xing, Yi

    2012-01-01

    Ultra-deep RNA sequencing has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We develop MATS (multivariate analysis of transcript splicing), a Bayesian statistical framework for flexible hypothesis testing of differential alternative splicing patterns on RNA-Seq data. MATS uses a multivariate uniform prior to model the between-sample correlation in exon splicing patterns, and a Markov chain Monte Carlo (MCMC) method coupled with a simulation-based adaptive sampling procedure to calculate the P-value and false discovery rate (FDR) of differential alternative splicing. Importantly, the MATS approach is applicable to almost any type of null hypotheses of interest, providing the flexibility to identify differential alternative splicing events that match a given user-defined pattern. We evaluated the performance of MATS using simulated and real RNA-Seq data sets. In the RNA-Seq analysis of alternative splicing events regulated by the epithelial-specific splicing factor ESRP1, we obtained a high RT–PCR validation rate of 86% for differential exon skipping events with a MATS FDR of <10%. Additionally, over the full list of RT–PCR tested exons, the MATS FDR estimates matched well with the experimental validation rate. Our results demonstrate that MATS is an effective and flexible approach for detecting differential alternative splicing from RNA-Seq data. PMID:22266656

  11. Differential network analysis reveals dysfunctional regulatory networks in gastric carcinogenesis

    PubMed Central

    Cao, Mu-Shui; Liu, Bing-Ya; Dai, Wen-Tao; Zhou, Wei-Xin; Li, Yi-Xue; Li, Yuan-Yuan

    2015-01-01

    Gastric Carcinoma is one of the most common cancers in the world. A large number of differentially expressed genes have been identified as being associated with gastric cancer progression, however, little is known about the underlying regulatory mechanisms. To address this problem, we developed a differential networking approach that is characterized by including a nascent methodology, differential coexpression analysis (DCEA), and two novel quantitative methods for differential regulation analysis. We first applied DCEA to a gene expression dataset of gastric normal mucosa, adenoma and carcinoma samples to identify gene interconnection changes during cancer progression, based on which we inferred normal, adenoma, and carcinoma-specific gene regulation networks by using linear regression model. It was observed that cancer genes and drug targets were enriched in each network. To investigate the dynamic changes of gene regulation during carcinogenesis, we then designed two quantitative methods to prioritize differentially regulated genes (DRGs) and gene pairs or links (DRLs) between adjacent stages. It was found that known cancer genes and drug targets are significantly higher ranked. The top 4% normal vs. adenoma DRGs (36 genes) and top 6% adenoma vs. carcinoma DRGs (56 genes) proved to be worthy of further investigation to explore their association with gastric cancer. Out of the 16 DRGs involved in two top-10 DRG lists of normal vs. adenoma and adenoma vs. carcinoma comparisons, 15 have been reported to be gastric cancer or cancer related. Based on our inferred differential networking information and known signaling pathways, we generated testable hypotheses on the roles of GATA6, ESRRG and their signaling pathways in gastric carcinogenesis. Compared with established approaches which build genome-scale GRNs, or sub-networks around differentially expressed genes, the present one proved to be better at enriching cancer genes and drug targets, and prioritizing

  12. Proteomic Characterization Reveals a Molecular Portrait of Nasopharyngeal Carcinoma Differentiation

    PubMed Central

    Xiao, Zhefeng; Li, Maoyu; Li, Guoqing; Fu, Ying; Peng, Fang; Chen, Yongheng; Chen, Zhuchu

    2017-01-01

    Nasopharyngeal carcinoma (NPC) is categorized into three different differentiated subtypes by World Health Organization (WHO). Based on an earlier comparative proteomic database of the three histological subtypes, the study was to deepen our understanding of molecular mechanisms associated with NPC differentiation through bio-information mining. Among the three subtypes were 194 differentially expressed proteins (DEPs) of 725 identified proteins. Two DEPs, heat shock protein family B (small) member 1 (HSPB1) and keratin 5 (KRT5), were validated in a series of NPC tissue samples by using immunohistochemistry. Quantified protein families including keratins, S100 proteins (S100s) and heat shock proteins exhibited characteristic expression alterations. Comparisons of predicted bio-function activation states among different subtypes, including formation of cellular protrusion, metastasis, cell death, and viral infections, were conducted. Canonical pathway analysis inferred that Rho GTPases related signaling pathways regulated the motility and invasion of dedifferentiated NPC. In conclusion, the study explored the proteomic characteristics of NPC differentiation, which could deepen our knowledge of NPC tumorigenesis and allow the development of novel targets of therapeutic and prognostic value in NPC. PMID:28367237

  13. Biophysical Characteristics Reveal Neural Stem Cell Differentiation Potential

    PubMed Central

    Mulhall, Hayley J.; Marchenko, Steve A.; Hoettges, Kai F.; Estrada, Laura C.; Lee, Abraham P.; Hughes, Michael P.; Flanagan, Lisa A.

    2011-01-01

    Background Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers. Methodology/Principal Findings We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates. Conclusions/Significance We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors. PMID:21980464

  14. Analysis of circadian pattern reveals tissue-specific alternative transcription in leptin signaling pathway

    PubMed Central

    Ptitsyn, Andrey A; Gimble, Jeffrey M

    2007-01-01

    Background It has been previously reported that most mammalian genes display a circadian oscillation in their baseline expression. Consequently, the phase and amplitude of each component of a signal transduction cascade has downstream consequences. Results Here, we report our analysis of alternative transcripts in the leptin signaling pathway which is responsible for the systemic regulation of macronutrient storage and energy balance. We focused on the circadian expression pattern of a critical component of the leptin signaling system, suppressor of cytokine signaling 3 (SOCS3). On an Affymetrix GeneChip 430A2 microarray, this gene is represented by three probe sets targeting different regions within the 3' end of the last exon. We demonstrate that in murine brown adipose tissue two downstream 3' probe sets experience circadian baseline oscillation in counter-phase to the upstream probe set. Such differences in expression patterns are a telltale sign of alternative splicing within the last exon of SOCS3. In contrast, all three probe sets oscillated in a common phase in murine liver and white adipose tissue. This suggests that the regulation of SOCS3 expression in brown fat is tissue specific. Another component of the signaling pathway, Janus kinase (JAK), is directly regulated by SOCS and has alternative transcript probe sets oscillating in counter-phase in a white adipose tissue specific manner. Conclusion We hypothesize that differential oscillation of alternative transcripts may provide a mechanism to maintain steady levels of expression in spite of circadian baseline variation. PMID:18047714

  15. Differential Network Analysis Reveals Genetic Effects on Catalepsy Modules

    PubMed Central

    Iancu, Ovidiu D.; Oberbeck, Denesa; Darakjian, Priscila; Kawane, Sunita; Erk, Jason; McWeeney, Shannon; Hitzemann, Robert

    2013-01-01

    We performed short-term bi-directional selective breeding for haloperidol-induced catalepsy, starting from three mouse populations of increasingly complex genetic structure: an F2 intercross, a heterogeneous stock (HS) formed by crossing four inbred strains (HS4) and a heterogeneous stock (HS-CC) formed from the inbred strain founders of the Collaborative Cross (CC). All three selections were successful, with large differences in haloperidol response emerging within three generations. Using a custom differential network analysis procedure, we found that gene coexpression patterns changed significantly; importantly, a number of these changes were concordant across genetic backgrounds. In contrast, absolute gene-expression changes were modest and not concordant across genetic backgrounds, in spite of the large and similar phenotypic differences. By inferring strain contributions from the parental lines, we are able to identify significant differences in allelic content between the selected lines concurrent with large changes in transcript connectivity. Importantly, this observation implies that genetic polymorphisms can affect transcript and module connectivity without large changes in absolute expression levels. We conclude that, in this case, selective breeding acts at the subnetwork level, with the same modules but not the same transcripts affected across the three selections. PMID:23555609

  16. Alternative Differential Identification Approaches for 2 Similar Bacilli Commonly Studied in Microbiology.

    ERIC Educational Resources Information Center

    Benathen, Isaiah A.

    1991-01-01

    Alternatives to the traditional unknown tests that permit a clear and unequivocal differential identification decision between Bacillus subtilis and Bacillus megaterium are presented. Plates of Phenylethyl Alcohol agar with Blood (PEAB), slants of Bile Esculin agar and plates of DNA agar are used. The materials, methods, results, and conclusions…

  17. A Review of Empirical Support for Differential Reinforcement of Alternative Behavior

    ERIC Educational Resources Information Center

    Petscher, Erin S.; Rey, Catalina; Bailey, Jon S.

    2009-01-01

    Differential reinforcement of alternative behavior (DRA) is one of the most common behavior analytic interventions used to decrease unwanted behavior. We reviewed the DRA literature from the past 30 years to identify the aspects that are thoroughly researched and those that would benefit from further emphasis. We found and coded 116 empirical…

  18. Comparing Main and Collateral Effects of Extinction and Differential Reinforcement of Alternative Behavior

    ERIC Educational Resources Information Center

    Petscher, Erin Seligson; Bailey, Jon S.

    2008-01-01

    This study evaluated the effects and collateral effects of extinction (EXT) and differential reinforcement of alternative behavior (DRA) interventions with inappropriate vocalizations and work refusal. Both interventions have been used frequently to reduce problem behaviors. The benefits of these interventions have been established yet may be…

  19. Effects of Treatment Integrity Failures during Differential Reinforcement of Alternative Behavior: A Translational Model

    ERIC Educational Resources Information Center

    Pipkin, Claire St. Peter; Vollmer, Timothy R.; Sloman, Kimberly N.

    2010-01-01

    Differential reinforcement of alternative behavior (DRA) is used frequently as a treatment for problem behavior. Previous studies on treatment integrity failures during DRA suggest that the intervention is robust, but research has not yet investigated the effects of different types of integrity failures. We examined the effects of two types of…

  20. Introducing Differential Equations Students to the Fredholm Alternative--In Staggered Doses

    ERIC Educational Resources Information Center

    Savoye, Philippe

    2011-01-01

    The development, in an introductory differential equations course, of boundary value problems in parallel with initial value problems and the Fredholm Alternative. Examples are provided of pairs of homogeneous and nonhomogeneous boundary value problems for which existence and uniqueness issues are considered jointly. How this heightens students'…

  1. SRSF10 Plays a Role in Myoblast Differentiation and Glucose Production via Regulation of Alternative Splicing.

    PubMed

    Wei, Ning; Cheng, Yuanming; Wang, Zhijia; Liu, Yuguo; Luo, Chunling; Liu, Lina; Chen, Linlin; Xie, Zhiqin; Lu, Yun; Feng, Ying

    2015-11-24

    Alternative splicing is a major mechanism of controlling gene expression and protein diversity in higher eukaryotes. We report that the splicing factor SRSF10 functions during striated muscle development, myoblast differentiation, and glucose production both in cells and in mice. A combination of RNA-sequencing and molecular analysis allowed us to identify muscle-specific splicing events controlled by SRSF10 that are critically involved in striated muscle development. Inclusion of alternative exons 16 and 17 of Lrrfip1 is a muscle-specific event that is activated by SRSF10 and essential for myoblast differentiation. On the other hand, in mouse primary hepatocytes, PGC1α is a key target of SRSF10 that regulates glucose production by fasting. SRSF10 represses inclusion of PGC1α exon 7a and facilitates the production of functional protein. The results highlight the biological significance of SRSF10 and regulated alternative splicing in vivo.

  2. Comparative Genomic Analysis Reveals Ecological Differentiation in the Genus Carnobacterium

    PubMed Central

    Iskandar, Christelle F.; Borges, Frédéric; Taminiau, Bernard; Daube, Georges; Zagorec, Monique; Remenant, Benoît; Leisner, Jørgen J.; Hansen, Martin A.; Sørensen, Søren J.; Mangavel, Cécile; Cailliez-Grimal, Catherine; Revol-Junelles, Anne-Marie

    2017-01-01

    Lactic acid bacteria (LAB) differ in their ability to colonize food and animal-associated habitats: while some species are specialized and colonize a limited number of habitats, other are generalist and are able to colonize multiple animal-linked habitats. In the current study, Carnobacterium was used as a model genus to elucidate the genetic basis of these colonization differences. Analyses of 16S rRNA gene meta-barcoding data showed that C. maltaromaticum followed by C. divergens are the most prevalent species in foods derived from animals (meat, fish, dairy products), and in the gut. According to phylogenetic analyses, these two animal-adapted species belong to one of two deeply branched lineages. The second lineage contains species isolated from habitats where contact with animal is rare. Genome analyses revealed that members of the animal-adapted lineage harbor a larger secretome than members of the other lineage. The predicted cell-surface proteome is highly diversified in C. maltaromaticum and C. divergens with genes involved in adaptation to the animal milieu such as those encoding biopolymer hydrolytic enzymes, a heme uptake system, and biopolymer-binding adhesins. These species also exhibit genes for gut adaptation and respiration. In contrast, Carnobacterium species belonging to the second lineage encode a poorly diversified cell-surface proteome, lack genes for gut adaptation and are unable to respire. These results shed light on the important genomics traits required for adaptation to animal-linked habitats in generalist Carnobacterium. PMID:28337181

  3. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct n-termini

    SciTech Connect

    Parra, Marilyn K.; Gee, Sherry L.; Koury, Mark J.; Mohandas, Narla; Conboy, John G.

    2003-03-25

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Three mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C, were identified far upstream of exon 2 in both mouse and human genomes; all three are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80kD 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135kD isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated upregulation of 80kD 4.1R during terminal erythroid differentiation. Together these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.

  4. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

    PubMed

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F

    2015-12-22

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system.

  5. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation

    PubMed Central

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F.

    2015-01-01

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. PMID:26703587

  6. A reporter mouse reveals lineage-specific and heterogeneous expression of IRF8 during lymphoid and myeloid cell differentiation1

    PubMed Central

    Wang, Hongsheng; Yan, Ming; Sun, Jiafang; Jain, Shweta; Yoshimi, Ryusuke; Abolfath, Sanaz Momben; Ozato, Keiko; Coleman, William G.; Ng, Ashley P.; Metcalf, Donald; DiRago, Ladina; Nutt, Stephen L.; Morse, Herbert C.

    2014-01-01

    The interferon regulatory factor family member 8 (IRF8) regulates differentiation of lymphoid and myeloid lineage cells by promoting or suppressing lineage-specific genes. How IRF8 promotes hematopoietic progenitors to commit to one lineage while preventing the development of alternative lineages is not known. Here we report an IRF8-EGFP fusion protein reporter mouse that revealed previously unrecognized patterns of IRF8 expression. Differentiation of hematopoietic stem cells into oligopotent progenitors is associated with progressive increases in IRF8-EGFP expression. However, significant induction of IRF8-EGFP is found in granulocyte-myeloid progenitors (GMPs) and the common lymphoid progenitors (CLPs) but not the megakaryocytic-erythroid progenitors. Surprisingly, IRF8-EGFP identifies three subsets of the seemingly homogeneous GMPs with an intermediate level of expression of EGFP defining bipotent progenitors that differentiation into either EGFPhi monocytic progenitors or EGFPlo granulocytic progenitors. Also surprisingly, IRF8-EGFP revealed a highly heterogeneous pre-pro-B population with a fluorescence intensity ranging from background to 4 orders above background. Interestingly, IRF8-EGFP readily distinguishes true B cell-committed (EGFPint) from those that are non-committed. Moreover, dendritic cell progenitors expressed extremely high levels of IRF8-EGFP. Taken together, the IRF8-EGFP reporter revealed previously unrecognized subsets with distinct developmental potentials in phenotypically well-defined oligopotent progenitors, providing new insights into the dynamic heterogeneity of developing hematopoietic progenitors. PMID:25024380

  7. Morphometric Differentiation Among Anastrepha fraterculus (Diptera: Tephritidae) Exploiting Sympatric Alternate Hosts.

    PubMed

    Gómez-Cendra, P V; Paulin, L E; Oroño, L; Ovruski, S M; Vilardi, J C

    2016-04-01

    Anastrepha fraterculus (Wiedemann) is currently considered a complex of cryptic species infesting fruits from Mexico to Argentina and represents an interesting biological model for evolutionary studies. Moreover, detecting and quantifying behavioral, morphological, and genetic differentiation among populations is also relevant to the application of environment-friendly control programs. Here, phenotypic differentiation among individuals coexisting in the wild in a Northern region of Argentina was unveiled and associated with host choice. Six morphometric traits were measured in sympatric flies exploiting three different host species. Phenotypic variation was shown to be host-dependent regardless of geographical or temporal overlap. Flies collected from synchronous alternate hosts (peach and walnut) differed from each other despite the lack of geographical isolation. By contrast, flies emerging from guavas that ripen about two months later than peach and walnut showed no significant differentiation in comparison to flies collected from walnuts, but they differ significantly from flies originating from peaches. This result is consistent with the hypothesis that the same population of flies shifts from walnuts to guavas throughout the year, whereas the population of flies that uses peaches as a host is probably exploiting other alternate hosts when peach availability decreases. Further research is needed to study the underlying mechanism. Results are consistent with previous molecular markers (inter-simple sequence repeat-ISSR) research on flies stemming from the same hosts and the same area, suggesting that differentiation among flies emerging from alternative hosts occurs at both genetic and phenotypic levels. The contribution of host preference in long-term genetic differentiation is discussed.

  8. Stimulus Fading and Response Elaboration in Differential Reinforcement for Alternative Behavior

    PubMed Central

    Schlichenmeyer, Kevin J.; Dube, William V.; Vargas-Irwin, Mariela

    2015-01-01

    A hallmark of applied behavior analysis is the development of function-based interventions for problem behavior. A widely recommended function-based intervention is differential reinforcement of alternative behavior (DRA), in which reinforcement is contingent upon socially acceptable alternatives to problem behavior (e.g., teaching communication skills). Typically, DRA is introduced under rich schedules of reinforcement. Although effective for initiating behavior change, rich schedules are often impractical in the natural setting. In this study, we evaluated the extent to which a stimulus fading program could be employed to elaborate alternative behavior (mands) in two individuals diagnosed with an Autism Spectrum Disorder. For both participants, problem behavior was reduced substantially upon implementation of the DRA procedure. Further, problem behavior rates remained low and mand rates decreased to more practical levels as the DRA behavioral requirements increased during the fading program. The fading approach demonstrated in this paper may be a useful component of intervention packages for clinicians. PMID:25844032

  9. Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation.

    PubMed

    Ozsolak, Fatih; Kapranov, Philipp; Foissac, Sylvain; Kim, Sang Woo; Fishilevich, Elane; Monaghan, A Paula; John, Bino; Milos, Patrice M

    2010-12-10

    The emerging discoveries on the link between polyadenylation and disease states underline the need to fully characterize genome-wide polyadenylation states. Here, we report comprehensive maps of global polyadenylation events in human and yeast generated using refinements to the Direct RNA Sequencing technology. This direct approach provides a quantitative view of genome-wide polyadenylation states in a strand-specific manner and requires only attomole RNA quantities. The polyadenylation profiles revealed an abundance of unannotated polyadenylation sites, alternative polyadenylation patterns, and regulatory element-associated poly(A)(+) RNAs. We observed differences in sequence composition surrounding canonical and noncanonical human polyadenylation sites, suggesting novel noncoding RNA-specific polyadenylation mechanisms in humans. Furthermore, we observed the correlation level between sense and antisense transcripts to depend on gene expression levels, supporting the view that overlapping transcription from opposite strands may play a regulatory role. Our data provide a comprehensive view of the polyadenylation state and overlapping transcription.

  10. Ex-vivo iTreg differentiation revisited: Convenient alternatives to existing strategies.

    PubMed

    Akkaya, Billur; Holstein, Amanda H; Isaac, Christopher; Maz, Mitra P; Glass, Deborah D; Shevach, Ethan M; Akkaya, Munir

    2017-02-01

    Ex-vivo differentiation of regulatory T cells (Tregs) from naïve CD4(+) T-cells has been widely used in immunological research. Isolation of a highly pure naïve T cell population is the key factor that determines the efficiency of subsequent Treg differentiation. Currently, this step relies mostly on FACS sorting, which is often costly, time consuming, and inconvenient. Alternatively, magnetic separation of T-cells can be performed; yet, available protocols fail to reach sort level purity and consequently result in low Treg differentiation efficiency. Here, we present the results of a comprehensive side-by-side comparison of various magnetic separation strategies and FACS sorting in multiple levels. Additionally, we propose a novel optimized custom made magnetic separation protocol, which not only yields sort level purity and Treg differentiation but also lowers the reagent costs up to 75% compared to the commercially available purification kits. The highly pure naïve CD4(+) T-cell population obtained by this versatile method can also be used for differentiation of other T-cell subsets; therefore this protocol may have broad applications in T-cell research.

  11. Alternative requirements for Vestigial, Scalloped, and Dmef2 during muscle differentiation in Drosophila melanogaster.

    PubMed

    Deng, Hua; Hughes, Sarah C; Bell, John B; Simmonds, Andrew J

    2009-01-01

    Vertebrate development requires the activity of the myocyte enhancer factor 2 (mef2) gene family for muscle cell specification and subsequent differentiation. Additionally, several muscle-specific functions of MEF2 family proteins require binding additional cofactors including members of the Transcription Enhancing Factor-1 (TEF-1) and Vestigial-like protein families. In Drosophila there is a single mef2 (Dmef2) gene as well single homologues of TEF-1 and vestigial-like, scalloped (sd), and vestigial (vg), respectively. To clarify the role(s) of these factors, we examined the requirements for Vg and Sd during Drosophila muscle specification. We found that both are required for muscle differentiation as loss of sd or vg leads to a reproducible loss of a subset of either cardiac or somatic muscle cells in developing embryos. This muscle requirement for Sd or Vg is cell specific, as ubiquitous overexpression of either or both of these proteins in muscle cells has a deleterious effect on muscle differentiation. Finally, using both in vitro and in vivo binding assays, we determined that Sd, Vg, and Dmef2 can interact directly. Thus, the muscle-specific phenotypes we have associated with Vg or Sd may be a consequence of alternative binding of Vg and/or Sd to Dmef2 forming alternative protein complexes that modify Dmef2 activity.

  12. Differential stimulation of the retina with subretinally injected exogenous neurotransmitter: A biomimetic alternative to electrical stimulation

    NASA Astrophysics Data System (ADS)

    Rountree, Corey M.; Inayat, Samsoon; Troy, John B.; Saggere, Laxman

    2016-12-01

    Subretinal stimulation of the retina with neurotransmitters, the normal means of conveying visual information, is a potentially better alternative to electrical stimulation widely used in current retinal prostheses for treating blindness from photoreceptor degenerative diseases. Yet, no subretinal electrical or chemical stimulation study has stimulated the OFF and ON pathways differentially through inner retinal activation. Here, we demonstrate the feasibility of differentially stimulating retinal ganglion cells (RGCs) through the inner nuclear layer of the retina with glutamate, a primary neurotransmitter chemical, in a biomimetic way. We show that controlled pulsatile delivery of glutamate into the subsurface of explanted wild-type rat retinas elicits highly localized simultaneous inhibitory and excitatory spike rate responses in OFF and ON RGCs. We also present the spatiotemporal characteristics of RGC responses to subretinally injected glutamate and the therapeutic stimulation parameters. Our findings could pave the way for future development of a neurotransmitter-based subretinal prosthesis offering more naturalistic vision and better visual acuity than electrical prostheses.

  13. Atorvastatin promotes human monocyte differentiation toward alternative M2 macrophages through p38 mitogen-activated protein kinase-dependent peroxisome proliferator-activated receptor γ activation.

    PubMed

    Zhang, Ou; Zhang, Jinying

    2015-05-01

    M1 and M2 macrophages are detectable in human atherosclerotic lesions, and M2 macrophages are present at locations distant from the lipid core in more stable zones of the plaque and appear to exert anti-inflammatory properties on M1 macrophages. Peroxisome proliferator-activated receptor (PPAR) γ promotes the differentiation of monocytes into anti-inflammatory M2 macrophages. Although both statins and PPARγ ligands have been reported to protect against the progression of atherosclerosis, no data are currently available regarding the implication of statins in the alternative differentiation of human monocytes. In the present study, we hypothesized that atorvastatin may exert novel effects to prime human monocytes toward an anti-inflammatory alternative M2 phenotype. To this aim, we first found that abundant M2 markers were expressed in human circulating monocytes after atorvastatin treatment. Moreover, atorvastatin was able to induce PPARγ expression and activation in human monocytes in vivo and in vitro, resulting in priming primary human monocytes differentiation into M2 macrophages with a more pronounced paracrine anti-inflammatory activity in M1 macrophages. Additional data with molecular approaches revealed that p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK) 1/2 activation was involved in atorvastatin-mediated PPARγ activation and enhanced alternative M2 macrophage phenotype. Collectively, our data demonstrated that atorvastatin promotes human monocyte differentiation toward alternative M2 macrophages via p38 MAPK-dependent PPARγ activation.

  14. Molecular Phylogeny of Sequenced Saccharomycetes Reveals Polyphyly of the Alternative Yeast Codon Usage

    PubMed Central

    Mühlhausen, Stefanie; Kollmar, Martin

    2014-01-01

    The universal genetic code defines the translation of nucleotide triplets, called codons, into amino acids. In many Saccharomycetes a unique alteration of this code affects the translation of the CUG codon, which is normally translated as leucine. Most of the species encoding CUG alternatively as serine belong to the Candida genus and were grouped into a so-called CTG clade. However, the “Candida genus” is not a monophyletic group and several Candida species are known to use the standard CUG translation. The codon identity could have been changed in a single branch, the ancestor of the Candida, or to several branches independently leading to a polyphyletic alternative yeast codon usage (AYCU). In order to resolve the monophyly or polyphyly of the AYCU, we performed a phylogenomics analysis of 26 motor and cytoskeletal proteins from 60 sequenced yeast species. By investigating the CUG codon positions with respect to sequence conservation at the respective alignment positions, we were able to unambiguously assign the standard code or AYCU. Quantitative analysis of the highly conserved leucine and serine alignment positions showed that 61.1% and 17% of the CUG codons coding for leucine and serine, respectively, are at highly conserved positions, whereas only 0.6% and 2.3% of the CUG codons, respectively, are at positions conserved in the respective other amino acid. Plotting the codon usage onto the phylogenetic tree revealed the polyphyly of the AYCU with Pachysolen tannophilus and the CTG clade branching independently within a time span of 30–100 Ma. PMID:25646540

  15. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining

    PubMed Central

    van Schendel, Robin; van Heteren, Jane; Welten, Richard; Tijsterman, Marcel

    2016-01-01

    For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. While inaccurate replication across damaged bases can explain the formation of single nucleotide variants, it remained unknown how DNA damage induces more severe genomic alterations. Here, we demonstrate for two of the most widely used mutagens, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs). This discovery allowed us to survey many thousands of available C. elegans deletion alleles to address the biology of this alternative end-joining repair mechanism. Analysis of ~7,000 deletion breakpoints and their cognate junctions reveals a distinct order of events. We found that nascent strands blocked at sites of DNA damage can engage in one or more cycles of primer extension using a more downstream located break end as a template. Resolution is accomplished when 3’ overhangs have matching ends. Our study provides a step-wise and versatile model for the in vivo mechanism of POLQ action, which explains the molecular nature of mutagen-induced deletion alleles. PMID:27755535

  16. Detection of recurrent alternative splicing switches in tumor samples reveals novel signatures of cancer

    PubMed Central

    Sebestyén, Endre; Zawisza, Michał; Eyras, Eduardo

    2015-01-01

    The determination of the alternative splicing isoforms expressed in cancer is fundamental for the development of tumor-specific molecular targets for prognosis and therapy, but it is hindered by the heterogeneity of tumors and the variability across patients. We developed a new computational method, robust to biological and technical variability, which identifies significant transcript isoform changes across multiple samples. We applied this method to more than 4000 samples from the The Cancer Genome Atlas project to obtain novel splicing signatures that are predictive for nine different cancer types, and find a specific signature for basal-like breast tumors involving the tumor-driver CTNND1. Additionally, our method identifies 244 isoform switches, for which the change occurs in the most abundant transcript. Some of these switches occur in known tumor drivers, including PPARG, CCND3, RALGDS, MITF, PRDM1, ABI1 and MYH11, for which the switch implies a change in the protein product. Moreover, some of the switches cannot be described with simple splicing events. Surprisingly, isoform switches are independent of somatic mutations, except for the tumor-suppressor FBLN2 and the oncogene MYH11. Our method reveals novel signatures of cancer in terms of transcript isoforms specifically expressed in tumors, providing novel potential molecular targets for prognosis and therapy. Data and software are available at: http://dx.doi.org/10.6084/m9.figshare.1061917 and https://bitbucket.org/regulatorygenomicsupf/iso-ktsp. PMID:25578962

  17. Detection of recurrent alternative splicing switches in tumor samples reveals novel signatures of cancer.

    PubMed

    Sebestyén, Endre; Zawisza, Michał; Eyras, Eduardo

    2015-02-18

    The determination of the alternative splicing isoforms expressed in cancer is fundamental for the development of tumor-specific molecular targets for prognosis and therapy, but it is hindered by the heterogeneity of tumors and the variability across patients. We developed a new computational method, robust to biological and technical variability, which identifies significant transcript isoform changes across multiple samples. We applied this method to more than 4000 samples from the The Cancer Genome Atlas project to obtain novel splicing signatures that are predictive for nine different cancer types, and find a specific signature for basal-like breast tumors involving the tumor-driver CTNND1. Additionally, our method identifies 244 isoform switches, for which the change occurs in the most abundant transcript. Some of these switches occur in known tumor drivers, including PPARG, CCND3, RALGDS, MITF, PRDM1, ABI1 and MYH11, for which the switch implies a change in the protein product. Moreover, some of the switches cannot be described with simple splicing events. Surprisingly, isoform switches are independent of somatic mutations, except for the tumor-suppressor FBLN2 and the oncogene MYH11. Our method reveals novel signatures of cancer in terms of transcript isoforms specifically expressed in tumors, providing novel potential molecular targets for prognosis and therapy. Data and software are available at: http://dx.doi.org/10.6084/m9.figshare.1061917 and https://bitbucket.org/regulatorygenomicsupf/iso-ktsp.

  18. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins.

    PubMed

    Huelga, Stephanie C; Vu, Anthony Q; Arnold, Justin D; Liang, Tiffany Y; Liu, Patrick P; Yan, Bernice Y; Donohue, John Paul; Shiue, Lily; Hoon, Shawn; Brenner, Sydney; Ares, Manuel; Yeo, Gene W

    2012-02-23

    Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

  19. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins

    PubMed Central

    Huelga, Stephanie C.; Vu, Anthony Q.; Arnold, Justin D.; Liang, Tiffany Y.; Liu, Patrick P.; Yan, Bernice Y.; Donohue, John Paul; Shiue, Lily; Hoon, Shawn; Brenner, Sydney; Ares, Manuel; Yeo, Gene W.

    2012-01-01

    SUMMARY Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells. PMID:22574288

  20. Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation.

    PubMed

    Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G; Xie, Jiuyong

    2012-09-01

    The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3' splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3' splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3' splice site usage.

  1. Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells.

    PubMed

    Liu, Jing; Lin, Jin; Huang, Lin-Feng; Huang, Bo; Xu, Yan-Mei; Li, Jing; Wang, Yan; Zhang, Jing; Yang, Wei-Ming; Min, Qing-Hua; Wang, Xiao-Zhong

    2015-09-01

    Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development.

  2. SES differentials in health by age and alternative indicators of SES.

    PubMed

    Robert, S; House, J S

    1996-08-01

    Despite the general persistence and even increase of strong socioeconomic status (SES) differentials in health in the United States, research suggests that SES differentials in health may diminish or become nonexistent at older ages. However, most research has used only limited measures of SES (e.g. education, income), and has not thoroughly investigated intra-elderly age differences in this trend. The current study investigates how SES differentials in health vary by age in the United States, using fairly detailed age categories (through ages 85+), and 2 alternative indicators (home ownership and liquid assets) of a major additional dimension of SES, financial assets, which may be especially important at older ages. We address (a) how strongly financial assets are associated with health, considered both alone and net of education and income; (b) if the health effects of financial assets vary by age; and, more specifically, (c) if their effects are especially pronounced in older age, again considered both alone and net of or relative to education and income. Results show that financial assets, especially liquid assets, considered both alone and net of education and income, are associated with health throughout adulthood and old age, at least until ages 85+. Furthermore, financial assets remain associated with health until quite late in life and become more important relative to education and income at older ages for some measures of health.

  3. Distribution and Differential Survival of Traditional and Alternative Indicators of Fecal Pollution at Freshwater Beaches.

    PubMed

    Cloutier, Danielle D; McLellan, Sandra L

    2017-02-15

    Alternative indicators have been developed that can be used to identify host sources of fecal pollution, yet little is known about how their distribution and fate compare to traditional indicators. Escherichia coli and enterococci were widely distributed at the six beaches studied and were detected in almost 95% of water samples (n = 422) and 100% of sand samples (n = 400). Berm sand contained the largest amount of E. coli (P < 0.01), whereas levels of enterococci were highest in the backshore (P < 0.01). E. coli and enterococci were the lowest in water, using a weight-to-volume comparison. The gull-associated Catellicoccus marimammalium (Gull2) marker was found in over 80% of water samples, regardless of E. coli levels, and in 25% of sand samples. Human-associated Bacteroides (HB) and Lachnospiraceae (Lachno2) were detected in only 2.4% of water samples collected under baseflow and post-rain conditions but produced a robust signal after a combined sewage overflow, despite low E. coli concentrations. Burdens of E. coli and enterococci in water and sand were disproportionately high in relation to alternative indicators when comparing environmental samples to source material. In microcosm studies, Gull2, HB, and Lachno2 quantitative PCR (qPCR) signals were reduced twice as quickly as those from E. coli and enterococci and approximately 20% faster than signals from culturable E. coli High concentrations of alternative indicators in source material illustrated their high sensitivity for the identification of fecal sources; however, differential survival and the potential for long-term persistence of traditional fecal indicators complicate the use of alternative indicator data to account for the levels of E. coli and enterococci in environmental samples.

  4. Two isoforms of TALDO1 generated by alternative translational initiation show differential nucleocytoplasmic distribution to regulate the global metabolic network

    PubMed Central

    Moriyama, Tetsuji; Tanaka, Shu; Nakayama, Yasumune; Fukumoto, Masahiro; Tsujimura, Kenji; Yamada, Kohji; Bamba, Takeshi; Yoneda, Yoshihiro; Fukusaki, Eiichiro; Oka, Masahiro

    2016-01-01

    Transaldolase 1 (TALDO1) is a rate-limiting enzyme involved in the pentose phosphate pathway, which is traditionally thought to occur in the cytoplasm. In this study, we found that the gene TALDO1 has two translational initiation sites, generating two isoforms that differ by the presence of the first 10 N-terminal amino acids. Notably, the long and short isoforms were differentially localised to the cell nucleus and cytoplasm, respectively. Pull-down and in vitro transport assays showed that the long isoform, unlike the short one, binds to importin α and is actively transported into the nucleus in an importin α/β-dependent manner, demonstrating that the 10 N-terminal amino acids are essential for its nuclear localisation. Additionally, we found that these two isoforms can form homo- and/or hetero-dimers with different localisation dynamics. A metabolite analysis revealed that the subcellular localisation of TALDO1 is not crucial for its activity in the pentose phosphate pathway. However, the expression of these two isoforms differentially affected the levels of various metabolites, including components of the tricarboxylic acid cycle, nucleotides, and sugars. These results demonstrate that the nucleocytoplasmic distribution of TALDO1, modulated via alternative translational initiation and dimer formation, plays an important role in a wide range of metabolic networks. PMID:27703206

  5. Saline Stress Alters the Temporal Patterns of Xylem Differentiation and Alternative Oxidase Expression in Developing Soybean Roots1

    PubMed Central

    Hilal, Mirna; Zenoff, Ana M.; Ponessa, Graciela; Moreno, Hortensia; Massa, Eddy M.

    1998-01-01

    We conducted a coordinated biochemical and morphometric analysis of the effect of saline conditions on the differentiation zone of developing soybean (Glycine max L.) roots. Between d 3 and d 14 for seedlings grown in control or NaCl-supplemented medium, we studied (a) the temporal evolution of the respiratory alternative oxidase (AOX) capacity in correlation with the expression and localization of AOX protein analyzed by tissue-print immunoblotting; (b) the temporal evolution and tissue localization of a peroxidase activity involved in lignification; and (c) the structural changes, visualized by light microscopy and quantified by image digitization. The results revealed that saline stress retards primary xylem differentiation. There is a corresponding delay in the temporal pattern of AOX expression, which is consistent with the xylem-specific localization of AOX protein and the idea that this enzyme is linked to xylem development. An NaCl-induced acceleration of the development of secondary xylem was also observed. However, the temporal pattern of a peroxidase activity localized in the primary and secondary xylem was unaltered by NaCl treatment. Thus, the NaCl-stressed root was specifically affected in the temporal patterns of AOX expression and xylem development. PMID:9625723

  6. Alternative splicing and gene duplication differentially shaped the regulation of isochorismate synthase in Populus and Arabidopsis

    PubMed Central

    Yuan, Yinan; Chung, Jeng-Der; Fu, Xueyan; Johnson, Virgil E.; Ranjan, Priya; Booth, Sarah L.; Harding, Scott A.; Tsai, Chung-Jui

    2009-01-01

    Isochorismate synthase (ICS) converts chorismate to isochorismate for the biosynthesis of phylloquinone, an essential cofactor for photosynthetic electron transport. ICS is also required for salicylic acid (SA) synthesis during Arabidopsis defense. In several other species, including Populus, SA is derived primarily from the phenylpropanoid pathway. We therefore sought to investigate ICS regulation in Populus to learn the extent of ICS involvement in SA synthesis and defense. Arabidopsis harbors duplicated AtICS genes that differ in their exon-intron structure, basal expression, and stress inducibility. In contrast, we found a single ICS gene in Populus and six other sequenced plant genomes, pointing to the AtICS duplication as a lineage-specific event. The Populus ICS encodes a functional plastidic enzyme, and was not responsive to stresses that stimulated phenylpropanoid accumulation. Populus ICS underwent extensive alternative splicing that was rare for the duplicated AtICSs. Sequencing of 184 RT-PCR Populus clones revealed 37 alternative splice variants, with normal transcripts representing ≈50% of the population. When expressed in Arabidopsis, Populus ICS again underwent alternative splicing, but did not produce normal transcripts to complement AtICS1 function. The splice-site sequences of Populus ICS are unusual, suggesting a causal link between junction sequence, alternative splicing, and ICS function. We propose that gene duplication and alternative splicing of ICS evolved independently in Arabidopsis and Populus in accordance with their distinct defense strategies. AtICS1 represents a divergent isoform for inducible SA synthesis during defense. Populus ICS primarily functions in phylloquinone biosynthesis, a process that can be sustained at low ICS transcript levels. PMID:19996170

  7. Gene Expression Profiling Reveals New Potential Players of Gonad Differentiation in the Chicken Embryo

    PubMed Central

    Carré, Gwenn-Aël; Couty, Isabelle; Hennequet-Antier, Christelle; Govoroun, Marina S.

    2011-01-01

    Background In birds as in mammals, a genetic switch determines whether the undifferentiated gonad develops into an ovary or a testis. However, understanding of the molecular pathway(s) involved in gonad differentiation is still incomplete. Methodology/Principal Findings With the aim of improving characterization of the molecular pathway(s) involved in gonad differentiation in the chicken embryo, we developed a large scale real time reverse transcription polymerase chain reaction approach on 110 selected genes for evaluation of their expression profiles during chicken gonad differentiation between days 5.5 and 19 of incubation. Hierarchical clustering analysis of the resulting datasets discriminated gene clusters expressed preferentially in the ovary or the testis, and/or at early or later periods of embryonic gonad development. Fitting a linear model and testing the comparisons of interest allowed the identification of new potential actors of gonad differentiation, such as Z-linked ADAMTS12, LOC427192 (corresponding to NIM1 protein) and CFC1, that are upregulated in the developing testis, and BMP3 and Z-linked ADAMTSL1, that are preferentially expressed in the developing ovary. Interestingly, the expression patterns of several members of the transforming growth factor β family were sexually dimorphic, with inhibin subunits upregulated in the testis, and bone morphogenetic protein subfamily members including BMP2, BMP3, BMP4 and BMP7, upregulated in the ovary. This study also highlighted several genes displaying asymmetric expression profiles such as GREM1 and BMP3 that are potentially involved in different aspects of gonad left-right asymmetry. Conclusion/Significance This study supports the overall conservation of vertebrate sex differentiation pathways but also reveals some particular feature of gene expression patterns during gonad development in the chicken. In particular, our study revealed new candidate genes which may be potential actors of chicken gonad

  8. Integrated live imaging and molecular profiling of embryoid bodies reveals a synchronized progression of early differentiation

    PubMed Central

    Boxman, Jonathan; Sagy, Naor; Achanta, Sirisha; Vadigepalli, Rajanikanth; Nachman, Iftach

    2016-01-01

    Embryonic stem cells can spontaneously differentiate into cell types of all germ layers within embryoid bodies (EBs) in a highly variable manner. Whether there exists an intrinsic differentiation program common to all EBs is unknown. Here, we present a novel combination of high-throughput live two-photon imaging and gene expression profiling to study early differentiation dynamics spontaneously occurring within developing EBs. Onset timing of Brachyury-GFP was highly variable across EBs, while the spatial patterns as well as the dynamics of mesendodermal progression following onset were remarkably similar. We therefore defined a ‘developmental clock’ using the Brachyury-GFP signal onset timing. Mapping snapshot gene expression measurements to this clock revealed their temporal trends, indicating that loss of pluripotency, formation of primitive streak and mesodermal lineage progression are synchronized in EBs. Exogenous activation of Wnt or BMP signaling accelerated the intrinsic clock. CHIR down-regulated Wnt3, allowing insights into dependency mechanisms between canonical Wnt signaling and multiple genes. Our findings reveal a developmental clock characteristic of an early differentiation program common to all EBs, further establishing them as an in vitro developmental model. PMID:27530599

  9. Epigenetic landscapes reveal transcription factors that regulate CD8(+) T cell differentiation.

    PubMed

    Yu, Bingfei; Zhang, Kai; Milner, J Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P; Pereira, Renata M; Crotty, Shane; Chang, John T; Pipkin, Matthew E; Wang, Wei; Goldrath, Ananda W

    2017-03-13

    Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8(+) T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8(+) T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8(+) T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8(+) T cell differentiation.

  10. Comparing main and collateral effects of extinction and differential reinforcement of alternative behavior.

    PubMed

    Petscher, Erin Seligson; Bailey, Jon S

    2008-07-01

    This study evaluated the effects and collateral effects of extinction (EXT) and differential reinforcement of alternative behavior (DRA) interventions with inappropriate vocalizations and work refusal. Both interventions have been used frequently to reduce problem behaviors. The benefits of these interventions have been established yet may be outweighed by the reported negative side effects that result. However, these collateral effects have rarely been measured or reported. DRA produced the most rapid reductions in behavior for 4 of the 5 participants. Other behaviors were measured for changes and showed that the desirable collateral effect of academic engagement tended to be higher during EXT than DRA. No evidence of EXT bursts was present with any participant, although EXT-induced aggression occurred with 1 participant.

  11. Differential fitness effects of immunocompetence and neighbourhood density in alternative female lizard morphs.

    PubMed

    Calsbeek, Ryan; Bonneaud, Camille; Smith, Thomas B

    2008-01-01

    1. A growing number of studies demonstrate that natural selection acts on traits important in whole animal performance and physiology. 2. Here we describe a heritable polymorphism in female dorsal pattern in the lizard Anolis sagrei (Dumeril & Bibron 1837). 3. Morphs did not differ in body size or habitat use (perch diameter), however, we show that the social environment, estimated by the number of female neighbours, had different selective effects on alternative morphs in nature. 4. We show that morphs displayed a significantly different immune response to phytohaemagglutinin. Furthermore, natural selection differentially acted on combinations of female morph and immunocompetence, favouring high levels of immune function in one morph and low levels of immune function in the other. 5. We discuss the possibility that morph-specific investments in life-history traits may lead to correlational selection between traits, even when those traits are likely to be determined by different genetic loci.

  12. Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields

    PubMed Central

    2011-01-01

    detected in the stimulation group, suggesting a connection with oxidative stress. Both differentiation factors and electrical stimulation improved hMSC differentiation potential to bone based on calcium deposition on day 28. Conclusions Electrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes. PMID:21269490

  13. Subfunctionalization of duplicate mitf genes associated with differential degeneration of alternative exons in fish.

    PubMed Central

    Altschmied, Joachim; Delfgaauw, Jacqueline; Wilde, Brigitta; Duschl, Jutta; Bouneau, Laurence; Volff, Jean-Nicolas; Schartl, Manfred

    2002-01-01

    The microphthalmia-associated transcription factor (MITF) exists in at least four isoforms. These are generated in higher vertebrates using alternative 5' exons and promoters from a single gene. Two separate genes (mitf-m and mitf-b), however, are present in different teleost fish species including the poeciliid Xiphophorus, the pufferfishes Fugu rubripes and Tetraodon nigroviridis, and the zebrafish Danio rerio. Fish proteins MITF-m and MITF-b correspond at both the structural and the expression levels to one particular bird/mammalian MITF isoform. In the teleost lineage subfunctionalization of mitf genes after duplication at least 100 million years ago is associated with the degeneration of alternative exons and, probably, regulatory elements and promoters. For example, a remnant of the first exon specific for MITF-m is detected within the pufferfish gene encoding MITF-b. Retracing the evolutionary history of mitf genes in vertebrates uncovered the differential recruitment of new introns specific for either the teleost or the bird/mammalian lineage. PMID:12019239

  14. Differential stimulation of the retina with subretinally injected exogenous neurotransmitter: A biomimetic alternative to electrical stimulation

    PubMed Central

    Rountree, Corey M.; Inayat, Samsoon; Troy, John B.; Saggere, Laxman

    2016-01-01

    Subretinal stimulation of the retina with neurotransmitters, the normal means of conveying visual information, is a potentially better alternative to electrical stimulation widely used in current retinal prostheses for treating blindness from photoreceptor degenerative diseases. Yet, no subretinal electrical or chemical stimulation study has stimulated the OFF and ON pathways differentially through inner retinal activation. Here, we demonstrate the feasibility of differentially stimulating retinal ganglion cells (RGCs) through the inner nuclear layer of the retina with glutamate, a primary neurotransmitter chemical, in a biomimetic way. We show that controlled pulsatile delivery of glutamate into the subsurface of explanted wild-type rat retinas elicits highly localized simultaneous inhibitory and excitatory spike rate responses in OFF and ON RGCs. We also present the spatiotemporal characteristics of RGC responses to subretinally injected glutamate and the therapeutic stimulation parameters. Our findings could pave the way for future development of a neurotransmitter-based subretinal prosthesis offering more naturalistic vision and better visual acuity than electrical prostheses. PMID:27929043

  15. Comparative proteomics reveals a significant bias toward alternative protein isoforms with conserved structure and function.

    PubMed

    Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L

    2012-09-01

    Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and

  16. Near-field deformation from the El Mayor-Cucapah earthquake revealed by differential LIDAR.

    PubMed

    Oskin, Michael E; Arrowsmith, J Ramon; Hinojosa Corona, Alejandro; Elliott, Austin J; Fletcher, John M; Fielding, Eric J; Gold, Peter O; Gonzalez Garcia, J Javier; Hudnut, Ken W; Liu-Zeng, Jing; Teran, Orlando J

    2012-02-10

    Large [moment magnitude (M(w)) ≥ 7] continental earthquakes often generate complex, multifault ruptures linked by enigmatic zones of distributed deformation. Here, we report the collection and results of a high-resolution (≥nine returns per square meter) airborne light detection and ranging (LIDAR) topographic survey of the 2010 M(w) 7.2 El Mayor-Cucapah earthquake that produced a 120-kilometer-long multifault rupture through northernmost Baja California, Mexico. This differential LIDAR survey completely captures an earthquake surface rupture in a sparsely vegetated region with pre-earthquake lower-resolution (5-meter-pixel) LIDAR data. The postevent survey reveals numerous surface ruptures, including previously undocumented blind faults within thick sediments of the Colorado River delta. Differential elevation changes show distributed, kilometer-scale bending strains as large as ~10(3) microstrains in response to slip along discontinuous faults cutting crystalline bedrock of the Sierra Cucapah.

  17. Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.

    PubMed

    Bell-Temin, Harris; Culver-Cochran, Ashley E; Chaput, Dale; Carlson, Christina M; Kuehl, Melanie; Burkhardt, Brant R; Bickford, Paula C; Liu, Bin; Stevens, Stanley M

    2015-12-01

    Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.

  18. Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics*

    PubMed Central

    Bell-Temin, Harris; Culver-Cochran, Ashley E.; Chaput, Dale; Carlson, Christina M.; Kuehl, Melanie; Burkhardt, Brant R.; Bickford, Paula C.; Liu, Bin; Stevens, Stanley M.

    2015-01-01

    Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells. PMID:26424600

  19. Genetic differentiation within Eriochoir sinensis (milne, edwards) revealed by allozyme and RAPD

    NASA Astrophysics Data System (ADS)

    Cui, Zhao-Xia; Xiang, Jian-Hai; Song, Lin-Sheng; Zhou, Ling-Hua; Shi, Wei-Liang

    2000-09-01

    We analyzed 17 allozymes, and 20 primers in order to detect the genetic differentiation between commercial populations (Changjiang River, Liaohe River) of Eriochoir sinensis. Ten allozymes (LDH, MDH, ME, IDH, EST, ALP, AAT, CTL, POD, SOD) showed 21 loci by vertically discontinuos buffer system polyacrylamide gel electrophoresis. RAPD profiles generated by 12 ten-base primers showed 63 loci. The percentage of polymorphic loci and the expected heterozygosity obtained by using allozyme analysis were lower than those obtained by RAPD. The index of similarity between these two populations were 0.955 and 0.932 as revealed by allozyme analysis and RAPD technology. There was gene flow between the above populations.

  20. Immature monocytes recruited to the ischemic mouse brain differentiate into macrophages with features of alternative activation.

    PubMed

    Miró-Mur, Francesc; Pérez-de-Puig, Isabel; Ferrer-Ferrer, Maura; Urra, Xabier; Justicia, Carles; Chamorro, Angel; Planas, Anna M

    2016-03-01

    Acute stroke induces a local inflammatory reaction causing leukocyte infiltration. Circulating monocytes are recruited to the ischemic brain and become tissue macrophages morphologically indistinguishable from reactive microglia. However, monocytes are a heterogeneous population of cells with different functions. Herein, we investigated the infiltration and fate of the monocyte subsets in a mouse model of focal brain ischemia by permanent occlusion of the distal portion of the middle cerebral artery. We separated two main subtypes of CD11b(hi) monocytes according to their expression of the surface markers Ly6C and CD43. Using adoptive transfer of reporter monocytes and monocyte depletion, we identified the pro-inflammatory Ly6C(hi)CD43(lo)CCR2(+) subset as the predominant monocytes recruited to the ischemic tissue. Monocytes were seen in the leptomeninges from where they entered the cortex along the penetrating arterioles. Four days post-ischemia, they had invaded the infarcted core, where they were often located adjacent to blood vessels. At this time, Iba-1(-) and Iba-1(+) cells in the ischemic tissue incorporated BrdU, but BrdU incorporation was rare in the reporter monocytes. The monocyte phenotype progressively changed by down-regulating Ly6C, up-regulating F4/80, expressing low or intermediate levels of Iba-1, and developing macrophage morphology. Moreover, monocytes progressively acquired the expression of typical markers of alternatively activated macrophages, like arginase-1 and YM-1. Collectively, the results show that stroke mobilized immature pro-inflammatory Ly6C(hi)CD43(lo) monocytes that acutely infiltrated the ischemic tissue reaching the core of the lesion. Monocytes differentiated to macrophages with features of alternative activation suggesting possible roles in tissue repair during the sub-acute phase of stroke.

  1. Alternatively spliced myeloid differentiation protein-2 inhibits TLR4-mediated lung inflammation.

    PubMed

    Tumurkhuu, Gantsetseg; Dagvadorj, Jargalsaikhan; Jones, Heather D; Chen, Shuang; Shimada, Kenichi; Crother, Timothy R; Arditi, Moshe

    2015-02-15

    We previously identified a novel alternatively spliced isoform of human myeloid differentiation protein-2 (MD-2s) that competitively inhibits binding of MD-2 to TLR4 in vitro. In this study, we investigated the protective role of MD-2s in LPS-induced acute lung injury by delivering intratracheally an adenovirus construct that expressed MD-2s (Ad-MD-2s). After adenovirus-mediated gene transfer, MD-2s was strongly expressed in lung epithelial cells and readily detected in bronchoalveolar lavage fluid. Compared to adenovirus serotype 5 containing an empty vector lacking a transgene control mice, Ad-MD-2s delivery resulted in significantly less LPS-induced inflammation in the lungs, including less protein leakage, cell recruitment, and expression of proinflammatory cytokines and chemokines, such as IL-6, keratinocyte chemoattractant, and MIP-2. Bronchoalveolar lavage fluid from Ad-MD-2s mice transferred into lungs of naive mice before intratracheal LPS challenge diminished proinflammatory cytokine levels. As house dust mite (HDM) sensitization is dependent on TLR4 and HDM Der p 2, a structural homolog of MD-2, we also investigated the effect of MD-2s on HDM-induced allergic airway inflammation. Ad-MD-2s given before HDM sensitization significantly inhibited subsequent allergic airway inflammation after HDM challenge, including reductions in eosinophils, goblet cell hyperplasia, and IL-5 levels. Our study indicates that the alternatively spliced short isoform of human MD-2 could be a potential therapeutic candidate to treat human diseases induced or exacerbated by TLR4 signaling, such as Gram-negative bacterial endotoxin-induced lung injury and HDM-triggered allergic lung inflammation.

  2. Alternatively spliced myeloid differentiation protein-2 (MD-2s) protein inhibits TLR4-mediated lung inflammation

    PubMed Central

    Tumurkhuu, Gantsetseg; Dagvadorj, Jargalsaikhan; Jones, Heather D.; Chen, Shuang; Shimada, Kenichi; Crother, Timothy R.; Arditi, Moshe

    2014-01-01

    We previously identified a novel alternatively spliced isoform of human myeloid differentiation protein-2 (MD-2s) that competitively inhibits binding of MD-2 to TLR4 in vitro. Here we investigated the protective role of MD-2s in LPS-induced acute lung injury by delivering intracheally (i.t.) an adenovirus construct that expressed MD-2s (Ad-MD-2s). After adenovirus-mediated gene transfer, MD-2s was strongly expressed in lung epithelial cells and readily detected in bronchoalveolar lavage fluid (BALF). Compared to Ad-EV control mice, Ad-MD-2s delivery resulted in significantly less LPS-induced inflammation in the lungs, including less protein leakage, cell recruitment, and expression of proinflammatory cytokines and chemokines, such as IL-6, KC, and MIP-2. BALF from Ad-MD-2s mice transferred into lungs of naive mice before i.t. LPS challenge diminished pro-inflammatory cytokine levels. As house dust mite (HDM) sensitization is dependent on TLR4 and HDM Der p 2, a structural homolog of MD-2, we also investigated the effect of MD-2s on house dust mite (HDM)-induced allergic airway inflammation. Ad-MD-2s given before HDM sensitization significantly inhibited subsequent allergic airway inflammation after HDM challenge, including reductions in eosinophils, goblet cell hyperplasia, and IL-5 levels. Our study indicates that the alternatively spliced short isoform of human MD-2 could be a potential therapeutic candidate to treat human diseases induced or exacerbated by TLR4 signaling, such as Gram-negative bacterial endotoxin-induced lung injury and house dust mite-triggered allergic lung inflammation. PMID:25576596

  3. Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus

    PubMed Central

    Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S.

    2016-01-01

    Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

  4. Diversity of sharp-wave–ripple LFP signatures reveals differentiated brain-wide dynamical events

    PubMed Central

    Ramirez-Villegas, Juan F.; Logothetis, Nikos K.; Besserve, Michel

    2015-01-01

    Sharp-wave–ripple (SPW-R) complexes are believed to mediate memory reactivation, transfer, and consolidation. However, their underlying neuronal dynamics at multiple scales remains poorly understood. Using concurrent hippocampal local field potential (LFP) recordings and functional MRI (fMRI), we study local changes in neuronal activity during SPW-R episodes and their brain-wide correlates. Analysis of the temporal alignment between SPW and ripple components reveals well-differentiated SPW-R subtypes in the CA1 LFP. SPW-R–triggered fMRI maps show that ripples aligned to the positive peak of their SPWs have enhanced neocortical metabolic up-regulation. In contrast, ripples occurring at the trough of their SPWs relate to weaker neocortical up-regulation and absent subcortical down-regulation, indicating differentiated involvement of neuromodulatory pathways in the ripple phenomenon mediated by long-range interactions. To our knowledge, this study provides the first evidence for the existence of SPW-R subtypes with differentiated CA1 activity and metabolic correlates in related brain areas, possibly serving different memory functions. PMID:26540729

  5. Characterization of an electrochemical mercury sensor using alternating current, cyclic, square wave and differential pulse voltammetry.

    PubMed

    Guerreiro, Gabriela V; Zaitouna, Anita J; Lai, Rebecca Y

    2014-01-31

    Here we report the characterization of an electrochemical mercury (Hg(2+)) sensor constructed with a methylene blue (MB)-modified and thymine-containing linear DNA probe. Similar to the linear probe electrochemical DNA sensor, the resultant sensor behaved as a "signal-off" sensor in alternating current voltammetry and cyclic voltammetry. However, depending on the applied frequency or pulse width, the sensor can behave as either a "signal-off" or "signal-on" sensor in square wave voltammetry (SWV) and differential pulse voltammetry (DPV). In SWV, the sensor showed "signal-on" behavior at low frequencies and "signal-off" behavior at high frequencies. In DPV, the sensor showed "signal-off" behavior at short pulse widths and "signal-on" behavior at long pulse widths. Independent of the sensor interrogation technique, the limit of detection was found to be 10nM, with a linear dynamic range between 10nM and 500nM. In addition, the sensor responded to Hg(2+) rather rapidly; majority of the signal change occurred in <20min. Overall, the sensor retains all the characteristics of this class of sensors; it is reagentless, reusable, sensitive, specific and selective. This study also highlights the feasibility of using a MB-modified probe for real-time sensing of Hg(2+), which has not been previously reported. More importantly, the observed "switching" behavior in SWV and DPV is potentially generalizable and should be applicable to most sensors in this class of dynamics-based electrochemical biosensors.

  6. Alternative robust estimators for autoregressive models with outliers using differential evolution algorithm

    NASA Astrophysics Data System (ADS)

    Addawe, Rizavel C.; Addawe, Joel M.; Magadia, Joselito C.

    2016-11-01

    The Least Squares (LS), Least Median Squares (LMdS), Reweighted Least Squares (RLS) and Trimmed Least Squares (TLS) estimators are used to obtain parameter estimates of AR models using DE algorithm. The empirical study indicated that, the RLS estimator seems to be very reasonable because of having smaller root mean square error (RMSE), particularly for the Gaussian AR(1) process with unknown drift and additive outliers. Moreover, while LS performs well on shorter processes with less percentage and smaller magnitude of additive outliers (AOS); RLS and TLS compare favorably with respect to LS for longer AR processes. Thus, this study recommends the Reweighted Least Squares estimator as an alternative to the LS estimator in the case of autoregressive processes with additive outliers. The experiment also demonstrates that Differential Evolution (DE) algorithm obtains optimal solutions for fitting first-order autoregressive processes with outliers using the estimators. At the request of all authors of the paper, and with the agreement of the Proceedings Editor, an updated version of this article was published on 15 December 2016. The original version supplied to AIP Publishing contained errors in some of the mathematical equations and in Table 2. The errors have been corrected in the updated and re-published article.

  7. Alternative inclusion of fibroblast growth factor receptor 2 exon IIIc in Dunning prostate tumors reveals unexpected epithelial mesenchymal plasticity.

    PubMed

    Oltean, Sebastian; Sorg, Brian S; Albrecht, Todd; Bonano, Vivian I; Brazas, Robert M; Dewhirst, Mark W; Garcia-Blanco, Mariano A

    2006-09-19

    In epithelial cells, alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells, the same process results in the synthesis of FGFR2(IIIc). Expression of the FGFR2(IIIc) isoform during prostate tumor progression suggests a disruption of the epithelial character of these tumors. To visualize the use of FGFR2 exon IIIc in prostate AT3 tumors in syngeneic rats, we constructed minigene constructs that report on alternative splicing. Imaging these alternative splicing decisions revealed unexpected mesenchymal-epithelial transitions in these primary tumors. These transitions were observed more frequently where tumor cells were in contact with stroma. Indeed, these transitions were frequently observed among lung micrometastases in the organ parenchyma and immediately adjacent to blood vessels. Our data suggest an unforeseen relationship between epithelial mesenchymal plasticity and malignant fitness.

  8. Fair Pairs and Three Part Praise--Developing the Sustained Use of Differential Reinforcement of Alternative Behaviour

    ERIC Educational Resources Information Center

    Williams, Hugh

    2012-01-01

    A training course of two after-school sessions was run for teaching assistants (TAs) in a UK inner city primary school. The subject of the training was classroom use of a version of differential reinforcement of alternative behaviours (DRAs) known as Fair Pairs. The training introduced the concept of "Three Part Praise" within Fair…

  9. Teacher Implementation of Trial-Based Functional Analysis and Differential Reinforcement of Alternative Behavior for Students with Challenging Behavior

    ERIC Educational Resources Information Center

    Flynn, Susan D.; Lo, Ya-yu

    2016-01-01

    The purpose of this study was to examine the effects of a training package on three middle school special education teachers' accurate implementation of trial-based functional analysis (TBFA) and differential reinforcement of alternative behavior (DRA) with their students with autism spectrum disorders or emotional and behavioral disorders in the…

  10. Differential Reinforcement of Alternative Behavior Increases Resistance to Extinction: Clinical Demonstration, Animal Modeling, and Clinical Test of One Solution

    ERIC Educational Resources Information Center

    Mace, F. Charles; McComas, Jennifer J.; Mauro, Benjamin C.; Progar, Patrick R.; Taylor, Bridget; Ervin, Ruth; Zangrillo, Amanda N.

    2010-01-01

    Basic research with pigeons on behavioral momentum suggests that differential reinforcement of alternative behavior (DRA) can increase the resistance of target behavior to change. This finding suggests that clinical applications of DRA may inadvertently increase the persistence of target behavior even as it decreases its frequency. We conducted…

  11. Adult Human Mesenchymal Stem Cell Differentiation at the Cell Population and Single-Cell Levels Under Alternating Electric Current

    PubMed Central

    Wechsler, Marissa E.; Hermann, Brian P.

    2016-01-01

    Mesenchymal stem cells, precursors that can differentiate into osteoblasts, chondrocytes, and adipocytes, have tremendous potential for derivation of cells with specific (e.g., osteogenic) phenotypes for tissue engineering and tissue regeneration applications. To date, the predominant strategy to achieve directed differentiation of MSCs into osteoblasts was to recapitulate the normal developmental ontogeny of osteoblasts using growth factors (e.g., bone morphogenetic proteins). In contrast, the effects of biophysical stimuli alone on such outcomes remain, at best, partially understood. This in vitro study examined and optimized the effects of alternating electric current alone on the differentiation of adult human mesenchymal stem cells (hMSCs) at the cell population and single-cell levels. hMSCs, cultured on flat, indium-tin-oxide-coated glass in the absence of supplemented exogenous growth factors were exposed to alternating electric current (5–40 μA, 5–10 Hz frequency, sinusoidal waveform), for 1–24 h daily for up to 21 consecutive days. Compared to results obtained from the respective controls, hMSC populations exposed to the alternating electric current alone (in the absence of exogenous growth factors) expressed genes at various stages of differentiation (specifically, TAZ, Runx-2, Osterix, Osteopontin, and Osteocalcin). Optimal osteogenic differentiation was achieved when hMSCs were exposed to a 10 μA, 10 Hz alternating electric current for 6 h daily for up to 21 days. Exclusive osteodifferentiation was observed since genes for the chondrocyte (Collagen Type II) and adipocyte (FABP-4) lineages were not expressed under all conditions of the biophysical stimulus tested. Single cell mRNAs for 45 genes (indicative of hMSC differentiation) were monitored using Fluidigm Systems. Homogeneous expression of the early osteodifferentiation genes (specifically, TAZ and Runx-2) was observed in hMSCs exposed to the alternating electric current at 7 and

  12. Computational analysis of translational readthrough proteins in Drosophila and yeast reveals parallels to alternative splicing

    PubMed Central

    Pancsa, Rita; Macossay-Castillo, Mauricio; Kosol, Simone; Tompa, Peter

    2016-01-01

    In translational readthrough (TR) the ribosome continues extending the nascent protein beyond the first in-frame termination codon. Due to the lack of dedicated analyses of eukaryotic TR cases, the associated functional-evolutionary advantages are still unclear. Here, based on a variety of computational methods, we describe the structural and functional properties of previously proposed D. melanogaster and S. cerevisiae TR proteins and extensions. We found that in D. melanogaster TR affects long proteins in mainly regulatory roles. Their TR-extensions are structurally disordered and rich in binding motifs, which, together with their cell-type- and developmental stage-dependent inclusion, suggest that similarly to alternatively spliced exons they rewire cellular interaction networks in a temporally and spatially controlled manner. In contrast, yeast TR proteins are rather short and fulfil mainly housekeeping functions, like translation. Yeast extensions usually lack disorder and linear motifs, which precludes elucidating their functional relevance with sufficient confidence. Therefore we propose that by being much more restricted and by lacking clear functional hallmarks in yeast as opposed to fruit fly, TR shows remarkable parallels with alternative splicing. Additionally, the lack of conservation of TR extensions among orthologous TR proteins suggests that TR-mediated functions may be generally specific to lower taxonomic levels. PMID:27561673

  13. Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy

    PubMed Central

    Struck, Adam J.; Gupta, Riti; Farnsworth, Dylan R.; Mahady, Amy E.; Eichinger, Katy; Thornton, Charles A.; Wang, Eric T.; Berglund, J. Andrew

    2016-01-01

    Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM. PMID:27681373

  14. Alternative splicing of spleen tyrosine kinase differentially regulates colorectal cancer progression

    PubMed Central

    Ni, Beibei; Hu, Jun; Chen, Dianke; Li, Li; Chen, Daici; Wang, Jianping; Wang, Lei

    2016-01-01

    Spleen tyrosine kinase (SYK) has been reported as a potential tumor suppressor in colorectal cancer (CRC). However, the role of alternative splicing of SYK in carcinogenesis remains unclear. In the present study, SYK isoforms were overexpressed in the human CRC HCT 116 cell line using lentiviral expression vectors to investigate the biological functions of full length SYK [SYK(L)] and short form SYK [SYK(S)] in CRC. Real-time cellular analysis and the 5-ethynyl-2-deoxyuridine assay were used to detect the effects of SYK(L) and SYK(S) on cell proliferation. Cell cycle progression and migration were assessed via flow cytometry and Transwell assays, respectively. The results revealed that the recombinant lentivirus with SYK(L) overexpression significantly suppressed the proliferation and metastasis of CRC cells, while SYK(S) overexpression did not. In addition, MTS assays demonstrated that SYK(L) and SYK(S) increased the cellular sensitivity to 5-fluorouracil (5-FU), suggesting that SYK(L) and 5-FU produce a significant synergistic effect on CRC cell proliferation, while SYK(S) has an effect on modulating CRC 5-FU sensitivity. Furthermore, quantitative polymerase chain reaction results revealed that SYK(L) was downregulated in 69% of 26 pairs of CRC and adjacent non-cancerous tissues, whereas SYK(S) exhibited no significant differences between tumor and normal tissues. Overall, the present data provides evidence that SYK(L) is a tumor suppressor in CRC, and both SYK(L) and SYK(S) may serve as important predictors in the chemotherapeutic treatment of CRC. PMID:27602108

  15. PMD patient mutations reveal a long-distance intronic interaction that regulates PLP1/DM20 alternative splicing

    PubMed Central

    Taube, Jennifer R.; Sperle, Karen; Banser, Linda; Seeman, Pavel; Cavan, Barbra Charina V.; Garbern, James Y.; Hobson, Grace M.

    2014-01-01

    Alternative splicing of the proteolipid protein 1 gene (PLP1) produces two forms, PLP1 and DM20, due to alternative use of 5′ splice sites with the same acceptor site in intron 3. The PLP1 form predominates in central nervous system RNA. Mutations that reduce the ratio of PLP1 to DM20, whether mutant or normal protein is formed, result in the X-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). We investigated the ability of sequences throughout PLP1 intron 3 to regulate alternative splicing using a splicing minigene construct transfected into the oligodendrocyte cell line, Oli-neu. Our data reveal that the alternative splice of PLP1 is regulated by a long-distance interaction between two highly conserved elements that are separated by 581 bases within the 1071-base intron 3. Further, our data suggest that a base-pairing secondary structure forms between these two elements, and we demonstrate that mutations of either element designed to destabilize the secondary structure decreased the PLP1/DM20 ratio, while swap mutations designed to restore the structure brought the PLP1/DM20 ratio to near normal levels. Sequence analysis of intron 3 in families with clinical symptoms of PMD who did not have coding-region mutations revealed mutations that segregated with disease in three families. We showed that these patient mutations, which potentially destabilize the secondary structure, also reduced the PLP1/DM20 ratio. This is the first report of patient mutations causing disease by disruption of a long-distance intronic interaction controlling alternative splicing. This finding has important implications for molecular diagnostics of PMD. PMID:24890387

  16. Multivoxel patterns reveal functionally differentiated networks underlying auditory feedback processing of speech.

    PubMed

    Zheng, Zane Z; Vicente-Grabovetsky, Alejandro; MacDonald, Ewen N; Munhall, Kevin G; Cusack, Rhodri; Johnsrude, Ingrid S

    2013-03-06

    The everyday act of speaking involves the complex processes of speech motor control. An important component of control is monitoring, detection, and processing of errors when auditory feedback does not correspond to the intended motor gesture. Here we show, using fMRI and converging operations within a multivoxel pattern analysis framework, that this sensorimotor process is supported by functionally differentiated brain networks. During scanning, a real-time speech-tracking system was used to deliver two acoustically different types of distorted auditory feedback or unaltered feedback while human participants were vocalizing monosyllabic words, and to present the same auditory stimuli while participants were passively listening. Whole-brain analysis of neural-pattern similarity revealed three functional networks that were differentially sensitive to distorted auditory feedback during vocalization, compared with during passive listening. One network of regions appears to encode an "error signal" regardless of acoustic features of the error: this network, including right angular gyrus, right supplementary motor area, and bilateral cerebellum, yielded consistent neural patterns across acoustically different, distorted feedback types, only during articulation (not during passive listening). In contrast, a frontotemporal network appears sensitive to the speech features of auditory stimuli during passive listening; this preference for speech features was diminished when the same stimuli were presented as auditory concomitants of vocalization. A third network, showing a distinct functional pattern from the other two, appears to capture aspects of both neural response profiles. Together, our findings suggest that auditory feedback processing during speech motor control may rely on multiple, interactive, functionally differentiated neural systems.

  17. Genetic variability and differentiation of Caragana microphylla populations as revealed by RAPD markers.

    PubMed

    Chen, X H; Gao, Y B

    2011-09-01

    Genetic variability in random amplified polymorphic DNA (RAPD) was studied in 90 individuals of Caragana microphylla, an outcrossing perennial shrub species, from five natural populations sampled in Inner Mongolia steppe of China on a small scale. Nineteen selected primers were used to amplify DNA samples, and totally 225 bands were detected. The percentage of polymorphic bands within populations ranged form 58.22% to 63.56%, with an average of 60% at the population level and 71.11% at the species level, indicating relatively high genetic variations in C. microphylla species. Shannon's information index (I) and Nei's gene diversity (h) showed the similar trend with each other. According to the analysis of Nei's gene diversity, the percentage of genetic variation among populations was 7.13%, indicating a low level of genetic differentiation among populations. There existed a strong gene flow (Nm = 3.26) among populations. Although AMOVA analysis also revealed most variation was within populations (phi(ST) = 4.1%), a significant proportion was observed among populations (P<0.001) in the present study, suggesting genetic differentiation occurred among populations at a certain extent. Based on Mantel's tests and the results of previous studies, the genetic structure pattern of C. microphylla accorded with the isolation-by-distance model on a very large scale, however, on a small scale, the significant genetic differentiation among populations might be enhanced by the micro-environmental divergence among the sampling sites, rather than by geographic factors. Analysis of the genetic variations of C. microphylla populations provided useful information for the adaptive strategy of Caragana species.

  18. Genetic Tagging During Human Mesoderm Differentiation Reveals Tripotent Lateral Plate Mesodermal Progenitors.

    PubMed

    Chin, Chee Jia; Cooper, Aaron R; Lill, Georgia R; Evseenko, Denis; Zhu, Yuhua; He, Chong Bin; Casero, David; Pellegrini, Matteo; Kohn, Donald B; Crooks, Gay M

    2016-05-01

    Although clonal studies of lineage potential have been extensively applied to organ specific stem and progenitor cells, much less is known about the clonal origins of lineages formed from the germ layers in early embryogenesis. We applied lentiviral tagging followed by vector integration site analysis (VISA) with high-throughput sequencing to investigate the ontogeny of the hematopoietic, endothelial and mesenchymal lineages as they emerge from human embryonic mesoderm. In contrast to studies that have used VISA to track differentiation of self-renewing stem cell clones that amplify significantly over time, we focused on a population of progenitor clones with limited self-renewal capability. Our analyses uncovered the critical influence of sampling on the interpretation of lentiviral tag sharing, particularly among complex populations with minimal clonal duplication. By applying a quantitative framework to estimate the degree of undersampling we revealed the existence of tripotent mesodermal progenitors derived from pluripotent stem cells, and the subsequent bifurcation of their differentiation into bipotent endothelial/hematopoietic or endothelial/mesenchymal progenitors. Stem Cells 2016;34:1239-1250.

  19. Semantic Differential Scale Method Can Reveal Multi-Dimensional Aspects of Mind Perception.

    PubMed

    Takahashi, Hideyuki; Ban, Midori; Asada, Minoru

    2016-01-01

    As humans, we tend to perceive minds in both living and non-living entities, such as robots. From a questionnaire developed in a previous mind perception study, authors found that perceived minds could be located on two dimensions "experience" and "agency." This questionnaire allowed the assessment of how we perceive minds of various entities from a multi-dimensional point of view. In this questionnaire, subjects had to evaluate explicit mental capacities of target characters (e.g., capacity to feel hunger). However, we sometimes perceive minds in non-living entities, even though we cannot attribute these evidently biological capacities to the entity. In this study, we performed a large-scale web survey to assess mind perception by using the semantic differential scale method. We revealed that two mind dimensions "emotion" and "intelligence," respectively, corresponded to the two mind dimensions (experience and agency) proposed in a previous mind perception study. We did this without having to ask about specific mental capacities. We believe that the semantic differential scale is a useful method to assess the dimensions of mind perception especially for non-living entities that are hard to be attributed to biological capacities.

  20. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics.

    PubMed

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L; Huber, Steven C; Zhao, Youfu

    2013-02-21

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence.

  1. RNA sequencing reveals differentially expressed genes as potential diagnostic and prognostic indicators of gallbladder carcinoma

    PubMed Central

    Jiang, Mingming; Fang, Meng; Ji, Jun; Wang, Aihua; Wang, Mengmeng; Jiang, Xiaoqing; Gao, Chunfang

    2015-01-01

    Gallbladder carcinoma (GBC) is a rare tumor with a dismal survival rate overall. Hence, there is an urgent need for exploring more specific and sensitive biomarkers for the diagnosis and treatment of GBC. At first, amplified total RNAs from two paired GBC tumors and adjacent non-tumorous tissues (ANTTs) were subjected to RNA sequencing. 161 genes were identified differentially expressed between tumors and ANTTs. Functional enrichment analysis indicated that the up-regulated genes in tumor were primarily associated with signaling molecules and enzyme modulators, and mainly involved in cell cycles and pathways in cancer. Twelve differentially expressed genes (DEGs) were further confirmed in another independent cohort of 35 GBC patients. Expression levels of BIRC5, TK1, TNNT1 and MMP9 were found to be positively related to postoperative relapse. There was also a significant correlation between BIRC5 expression and tumor-node-metastasis (TNM) stage. Besides, we observed a positive correlation between serum CA19–9 concentration and the expression levels of TNNT1, MMP9 and CLIC3. Survival analysis revealed that GBC patients with high TK1 and MMP9 expression levels had worse prognosis. These identified DEGs might not only be promising biomarkers for GBC diagnosis and prognosis, but also expedite the discovery of novel therapeutic strategies. PMID:25970782

  2. Semantic Differential Scale Method Can Reveal Multi-Dimensional Aspects of Mind Perception

    PubMed Central

    Takahashi, Hideyuki; Ban, Midori; Asada, Minoru

    2016-01-01

    As humans, we tend to perceive minds in both living and non-living entities, such as robots. From a questionnaire developed in a previous mind perception study, authors found that perceived minds could be located on two dimensions “experience” and “agency.” This questionnaire allowed the assessment of how we perceive minds of various entities from a multi-dimensional point of view. In this questionnaire, subjects had to evaluate explicit mental capacities of target characters (e.g., capacity to feel hunger). However, we sometimes perceive minds in non-living entities, even though we cannot attribute these evidently biological capacities to the entity. In this study, we performed a large-scale web survey to assess mind perception by using the semantic differential scale method. We revealed that two mind dimensions “emotion” and “intelligence,” respectively, corresponded to the two mind dimensions (experience and agency) proposed in a previous mind perception study. We did this without having to ask about specific mental capacities. We believe that the semantic differential scale is a useful method to assess the dimensions of mind perception especially for non-living entities that are hard to be attributed to biological capacities. PMID:27853445

  3. Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis

    PubMed Central

    Marquez, Yamile; Brown, John W.S.; Simpson, Craig; Barta, Andrea; Kalyna, Maria

    2012-01-01

    Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. As very few genome-wide studies analyzing AS in plants are available, we have performed high-throughput sequencing of a normalized cDNA library which resulted in a high coverage transcriptome map of Arabidopsis. We detect ∼150,000 splice junctions derived mostly from typical plant introns, including an eightfold increase in the number of U12 introns (2069). Around 61% of multiexonic genes are alternatively spliced under normal growth conditions. Moreover, we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using high-resolution RT-PCR and Sanger sequencing. Intron retention (IR) is the most frequent AS event (∼40%), but many IRs have relatively low read coverage and are less well-represented in assembled transcripts. Additionally, ∼51% of Arabidopsis genes produce AS transcripts which do not involve IR. Therefore, the significance of IR in generating transcript diversity was generally overestimated in previous assessments. IR analysis allowed the identification of a large set of cryptic introns inside annotated coding exons. Importantly, a significant fraction of these cryptic introns are spliced out in frame, indicating a role in protein diversity. Furthermore, we show extensive AS coupled to nonsense-mediated decay in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, thus establishing a complex loop in AS regulation. We provide the most comprehensive analysis of AS to date which will serve as a valuable resource for the plant community to study transcriptome complexity and gene regulation. PMID:22391557

  4. Alternative splicing of U2AF1 reveals a shared repression mechanism for duplicated exons

    PubMed Central

    Kralovicova, Jana; Vorechovsky, Igor

    2017-01-01

    The auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF) facilitates branch point (BP) recognition and formation of lariat introns. The gene for the 35-kD subunit of U2AF gives rise to two protein isoforms (termed U2AF35a and U2AF35b) that are encoded by alternatively spliced exons 3 and Ab, respectively. The splicing recognition sequences of exon 3 are less favorable than exon Ab, yet U2AF35a expression is higher than U2AF35b across tissues. We show that U2AF35b repression is facilitated by weak, closely spaced BPs next to a long polypyrimidine tract of exon Ab. Each BP lacked canonical uridines at position -2 relative to the BP adenines, with efficient U2 base-pairing interactions predicted only for shifted registers reminiscent of programmed ribosomal frameshifting. The BP cluster was compensated by interactions involving unpaired cytosines in an upstream, EvoFold-predicted stem loop (termed ESL) that binds FUBP1/2. Exon Ab inclusion correlated with predicted free energies of mutant ESLs, suggesting that the ESL operates as a conserved rheostat between long inverted repeats upstream of each exon. The isoform-specific U2AF35 expression was U2AF65-dependent, required interactions between the U2AF-homology motif (UHM) and the α6 helix of U2AF35, and was fine-tuned by exon Ab/3 variants. Finally, we identify tandem homologous exons regulated by U2AF and show that their preferential responses to U2AF65-related proteins and SRSF3 are associated with unpaired pre-mRNA segments upstream of U2AF-repressed 3′ss. These results provide new insights into tissue-specific subfunctionalization of duplicated exons in vertebrate evolution and expand the repertoire of exon repression mechanisms that control alternative splicing. PMID:27566151

  5. Functional characterisation of an intron retaining K(+) transporter of barley reveals intron-mediated alternate splicing.

    PubMed

    Shahzad, K; Rauf, M; Ahmed, M; Malik, Z A; Habib, I; Ahmed, Z; Mahmood, K; Ali, R; Masmoudi, K; Lemtiri-Chlieh, F; Gehring, C; Berkowitz, G A; Saeed, N A

    2015-07-01

    Intron retention in transcripts and the presence of 5' and 3' splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K(+) transporter, HvHKT2;1, with stably retained introns from barley (Hordeum vulgare) in yeast (Saccharomyces cerevisiae), and transcript profiling in yeast and transgenic tobacco (Nicotiana tabacum) is presented. Expression of intron-retaining HvHKT2;1 cDNA (HvHKT2;1-i) in trk1, trk2 yeast strain defective in K(+) uptake restored growth in medium containing hygromycin in the presence of different concentrations of K(+) and mediated hypersensitivity to Na(+) . HvHKT2;1-i produces multiple transcripts via alternate splicing of two regular introns and three exons in different compositions. HKT isoforms with retained introns and exon skipping variants were detected in relative expression analysis of (i) HvHKT2;1-i in barley under native conditions, (ii) in transgenic tobacco plants constitutively expressing HvHKT2;1-i, and (iii) in trk1, trk2 yeast expressing HvHKT2;1-i under control of an inducible promoter. Mixed proportions of three HKT transcripts: HvHKT2;1-e (first exon region), HvHKT2;1-i1 (first intron) and HvHKT2;1-i2 (second intron) were observed. The variation in transcript accumulation in response to changing K(+) and Na(+) concentrations was observed in both heterologous and plant systems. These findings suggest a link between intron-retaining transcripts and different splice variants to ion homeostasis, and their possible role in salt stress.

  6. Exome analysis reveals differentially mutated gene signatures of stage, grade and subtype in breast cancers.

    PubMed

    Li, You; Wang, Xiaosheng; Vural, Suleyman; Mishra, Nitish K; Cowan, Kenneth H; Guda, Chittibabu

    2015-01-01

    Breast cancers exhibit highly heterogeneous molecular profiles. Although gene expression profiles have been used to predict the risks and prognostic outcomes of breast cancers, the high variability of gene expression limits its clinical application. In contrast, genetic mutation profiles would be more advantageous than gene expression profiles because genetic mutations can be stably detected and the mutational heterogeneity widely exists in breast cancer genomes. We analyzed 98 breast cancer whole exome samples that were sorted into three subtypes, two grades and two stages. The sum deleterious effect of all mutations in each gene was scored to identify differentially mutated genes (DMGs) for this case-control study. DMGs were corroborated using extensive published knowledge. Functional consequences of deleterious SNVs on protein structure and function were also investigated. Genes such as ERBB2, ESP8, PPP2R4, KIAA0922, SP4, CENPJ, PRCP and SELP that have been experimentally or clinically verified to be tightly associated with breast cancer prognosis are among the DMGs identified in this study. We also identified some genes such as ARL6IP5, RAET1E, and ANO7 that could be crucial for breast cancer development and prognosis. Further, SNVs such as rs1058808, rs2480452, rs61751507, rs79167802, rs11540666, and rs2229437 that potentially influence protein functions are observed at significantly different frequencies in different comparison groups. Protein structure modeling revealed that many non-synonymous SNVs have a deleterious effect on protein stability, structure and function. Mutational profiling at gene- and SNV-level revealed differential patterns within each breast cancer comparison group, and the gene signatures correlate with expected prognostic characteristics of breast cancer classes. Some of the genes and SNVs identified in this study show high promise and are worthy of further investigation by experimental studies.

  7. Exome Analysis Reveals Differentially Mutated Gene Signatures of Stage, Grade and Subtype in Breast Cancers

    PubMed Central

    Li, You; Wang, Xiaosheng; Vural, Suleyman; Mishra, Nitish K.; Cowan, Kenneth H.; Guda, Chittibabu

    2015-01-01

    Breast cancers exhibit highly heterogeneous molecular profiles. Although gene expression profiles have been used to predict the risks and prognostic outcomes of breast cancers, the high variability of gene expression limits its clinical application. In contrast, genetic mutation profiles would be more advantageous than gene expression profiles because genetic mutations can be stably detected and the mutational heterogeneity widely exists in breast cancer genomes. We analyzed 98 breast cancer whole exome samples that were sorted into three subtypes, two grades and two stages. The sum deleterious effect of all mutations in each gene was scored to identify differentially mutated genes (DMGs) for this case-control study. DMGs were corroborated using extensive published knowledge. Functional consequences of deleterious SNVs on protein structure and function were also investigated. Genes such as ERBB2, ESP8, PPP2R4, KIAA0922, SP4, CENPJ, PRCP and SELP that have been experimentally or clinically verified to be tightly associated with breast cancer prognosis are among the DMGs identified in this study. We also identified some genes such as ARL6IP5, RAET1E, and ANO7 that could be crucial for breast cancer development and prognosis. Further, SNVs such as rs1058808, rs2480452, rs61751507, rs79167802, rs11540666, and rs2229437 that potentially influence protein functions are observed at significantly different frequencies in different comparison groups. Protein structure modeling revealed that many non-synonymous SNVs have a deleterious effect on protein stability, structure and function. Mutational profiling at gene- and SNV-level revealed differential patterns within each breast cancer comparison group, and the gene signatures correlate with expected prognostic characteristics of breast cancer classes. Some of the genes and SNVs identified in this study show high promise and are worthy of further investigation by experimental studies. PMID:25803781

  8. Differential Gene Expression Reveals Candidate Genes for Drought Stress Response in Abies alba (Pinaceae).

    PubMed

    Behringer, David; Zimmermann, Heike; Ziegenhagen, Birgit; Liepelt, Sascha

    2015-01-01

    Increasing drought periods as a result of global climate change pose a threat to many tree species by possibly outpacing their adaptive capabilities. Revealing the genetic basis of drought stress response is therefore implemental for future conservation strategies and risk assessment. Access to informative genomic regions is however challenging, especially for conifers, partially due to their large genomes, which puts constraints on the feasibility of whole genome scans. Candidate genes offer a valuable tool to reduce the complexity of the analysis and the amount of sequencing work and costs. For this study we combined an improved drought stress phenotyping of needles via a novel terahertz water monitoring technique with Massive Analysis of cDNA Ends to identify candidate genes for drought stress response in European silver fir (Abies alba Mill.). A pooled cDNA library was constructed from the cotyledons of six drought stressed and six well-watered silver fir seedlings, respectively. Differential expression analyses of these libraries revealed 296 candidate genes for drought stress response in silver fir (247 up- and 49 down-regulated) of which a subset was validated by RT-qPCR of the twelve individual cotyledons. A majority of these genes code for currently uncharacterized proteins and hint on new genomic resources to be explored in conifers. Furthermore, we could show that some traditional reference genes from model plant species (GAPDH and eIF4A2) are not suitable for differential analysis and we propose a new reference gene, TPC1, for drought stress expression profiling in needles of conifer seedlings.

  9. Differential Gene Expression Reveals Candidate Genes for Drought Stress Response in Abies alba (Pinaceae)

    PubMed Central

    Ziegenhagen, Birgit; Liepelt, Sascha

    2015-01-01

    Increasing drought periods as a result of global climate change pose a threat to many tree species by possibly outpacing their adaptive capabilities. Revealing the genetic basis of drought stress response is therefore implemental for future conservation strategies and risk assessment. Access to informative genomic regions is however challenging, especially for conifers, partially due to their large genomes, which puts constraints on the feasibility of whole genome scans. Candidate genes offer a valuable tool to reduce the complexity of the analysis and the amount of sequencing work and costs. For this study we combined an improved drought stress phenotyping of needles via a novel terahertz water monitoring technique with Massive Analysis of cDNA Ends to identify candidate genes for drought stress response in European silver fir (Abies alba Mill.). A pooled cDNA library was constructed from the cotyledons of six drought stressed and six well-watered silver fir seedlings, respectively. Differential expression analyses of these libraries revealed 296 candidate genes for drought stress response in silver fir (247 up- and 49 down-regulated) of which a subset was validated by RT-qPCR of the twelve individual cotyledons. A majority of these genes code for currently uncharacterized proteins and hint on new genomic resources to be explored in conifers. Furthermore, we could show that some traditional reference genes from model plant species (GAPDH and eIF4A2) are not suitable for differential analysis and we propose a new reference gene, TPC1, for drought stress expression profiling in needles of conifer seedlings. PMID:25924061

  10. Genome-wide identification of Fas/CD95 alternative splicing regulators reveals links with iron homeostasis.

    PubMed

    Tejedor, J Ramón; Papasaikas, Panagiotis; Valcárcel, Juan

    2015-01-08

    Alternative splicing of Fas/CD95 exon 6 generates either a membrane-bound receptor that promotes, or a soluble isoform that inhibits, apoptosis. Using an automatized genome-wide siRNA screening for alternative splicing regulators of endogenous transcripts in mammalian cells, we identified 200 genes whose knockdown modulates the ratio between Fas/CD95 isoforms. These include classical splicing regulators; core spliceosome components; and factors implicated in transcription and chromatin remodeling, RNA transport, intracellular signaling, and metabolic control. Coherent effects of genes involved in iron homeostasis and pharmacological modulation of iron levels revealed a link between intracellular iron and Fas/CD95 exon 6 inclusion. A splicing regulatory network linked iron levels with reduced activity of the Zinc-finger-containing splicing regulator SRSF7, and in vivo and in vitro assays revealed that iron inhibits SRSF7 RNA binding. Our results uncover numerous links between cellular pathways and RNA processing and a mechanism by which iron homeostasis can influence alternative splicing.

  11. Differential spectral power alteration following acupuncture at different designated places revealed by magnetoencephalography

    NASA Astrophysics Data System (ADS)

    You, Youbo; Bai, Lijun; Dai, Ruwei; Xue, Ting; Zhong, Chongguang; Liu, Zhenyu; Wang, Hu; Feng, Yuanyuan; Wei, Wenjuan; Tian, Jie

    2012-03-01

    As an ancient therapeutic technique in Traditional Chinese Medicine, acupuncture has been used increasingly in modern society to treat a range of clinical conditions as an alternative and complementary therapy. However, acupoint specificity, lying at the core of acupuncture, still faces many controversies. Considering previous neuroimaging studies on acupuncture have mainly employed functional magnetic resonance imaging, which only measures the secondary effect of neural activity on cerebral metabolism and hemodynamics, in the current study, we adopted an electrophysiological measurement technique named magnetoencephalography (MEG) to measure the direct neural activity. 28 healthy college students were recruited in this study. We filtered MEG data into 5 consecutive frequency bands (delta, theta, alpha, beta and gamma band) and grouped 140 sensors into 10 main brain regions (left/right frontal, central, temporal, parietal and occipital regions). Fast Fourier Transformation (FFT) based spectral analysis approach was further performed to explore the differential band-limited power change patterns of acupuncture at Stomach Meridian 36 (ST36) using a nearby nonacupoint (NAP) as control condition. Significantly increased delta power and decreased alpha as well as beta power in bilateral frontal ROIs were observed following stimulation at ST36. Compared with ST36, decreased alpha power in left and right central, right parietal as well as right temporal ROIs were detected in NAP group. Our research results may provide additional evidence for acupoint specificity.

  12. Transcriptome analyses and differential gene expression in a non-model fish species with alternative mating tactics

    PubMed Central

    2014-01-01

    Background Social dominance is important for the reproductive success of males in many species. In the black-faced blenny (Tripterygion delaisi) during the reproductive season, some males change color and invest in nest making and defending a territory, whereas others do not change color and ‘sneak’ reproductions when females lay their eggs. Using RNAseq, we profiled differential gene expression between the brains of territorial males, sneaker males, and females to study the molecular signatures of male dimorphism. Results We found that more genes were differentially expressed between the two male phenotypes than between males and females, suggesting that during the reproductive period phenotypic plasticity is a more important factor in differential gene expression than sexual dimorphism. The territorial male overexpresses genes related to synaptic plasticity and the sneaker male overexpresses genes involved in differentiation and development. Conclusions Previously suggested candidate genes for social dominance in the context of alternative mating strategies seem to be predominantly species-specific. We present a list of novel genes which are differentially expressed in Tripterygion delaisi. This is the first genome-wide study for a molecular non-model species in the context of alternative mating strategies and provides essential information for further studies investigating the molecular basis of social dominance. PMID:24581002

  13. Quantitative Proteomics Reveals GIMAP Family Proteins 1 and 4 to Be Differentially Regulated during Human T Helper Cell Differentiation *S⃞

    PubMed Central

    Filén, Jan-Jonas; Filén, Sanna; Moulder, Robert; Tuomela, Soile; Ahlfors, Helena; West, Anne; Kouvonen, Petri; Kantola, Suvi; Björkman, Mari; Katajamaa, Mikko; Rasool, Omid; Nyman, Tuula A.; Lahesmaa, Riitta

    2009-01-01

    T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet- and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4+ cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GIMAP4 genes are up-regulated by IL-12 and other Th1 differentiation-inducing cytokines in cells induced to differentiate toward Th1 lineage and down-regulated by IL-4 in cells induced to Th2. Our results indicate that the GIMAP (GTPase of the immunity-associated protein) family of proteins is differentially regulated during Th cell differentiation. PMID:18701445

  14. Sister Dehalobacter Genomes Reveal Specialization in Organohalide Respiration and Recent Strain Differentiation Likely Driven by Chlorinated Substrates

    SciTech Connect

    Tang, Shuiquan; Wang, Po Hsiang; Higgins, Steven A.; Löffler, Frank E.; Edwards, Elizabeth A.

    2016-02-12

    Here we report that the genomes of two closely related Dehalobacter strains (strain CF and strain DCA) were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF), 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA). The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA) and strain DCA (that dechlorinates 1,1-DCA) each contain 17 putative reductive dehalogenase homologous (rdh) genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB), and to the genomes of Dehalococcoides mccartyi strain 195 and Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and >99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1) and corrinoid biosynthesis pathways (strains E1 and PER-K23). The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to D. mccartyi, a complete

  15. Sister Dehalobacter Genomes Reveal Specialization in Organohalide Respiration and Recent Strain Differentiation Likely Driven by Chlorinated Substrates

    DOE PAGES

    Tang, Shuiquan; Wang, Po Hsiang; Higgins, Steven A.; ...

    2016-02-12

    Here we report that the genomes of two closely related Dehalobacter strains (strain CF and strain DCA) were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF), 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA). The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA) and strain DCA (that dechlorinates 1,1-DCA) each contain 17 putative reductive dehalogenase homologous (rdh) genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB), and to the genomes of Dehalococcoides mccartyi strain 195 andmore » Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and >99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1) and corrinoid biosynthesis pathways (strains E1 and PER-K23). The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to D. mccartyi, a

  16. Sister Dehalobacter Genomes Reveal Specialization in Organohalide Respiration and Recent Strain Differentiation Likely Driven by Chlorinated Substrates

    PubMed Central

    Tang, Shuiquan; Wang, Po Hsiang; Higgins, Steven A.; Löffler, Frank E.; Edwards, Elizabeth A.

    2016-01-01

    The genomes of two closely related Dehalobacter strains (strain CF and strain DCA) were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF), 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA). The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA) and strain DCA (that dechlorinates 1,1-DCA) each contain 17 putative reductive dehalogenase homologous (rdh) genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB), and to the genomes of Dehalococcoides mccartyi strain 195 and Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and >99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1) and corrinoid biosynthesis pathways (strains E1 and PER-K23). The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to D. mccartyi, a complete heme biosynthesis

  17. Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing

    SciTech Connect

    Du, Kejun; Chen, Yaoming; Dai, Zongming; Bi, Yuan; Cai, Tongjian; Hou, Lichao; Chai, Yubo; Song, Qinghe; Chen, Sumin; Luo, Wenjing; Chen, Jingyuan

    2010-01-01

    Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. (DQ316984) and (DQ320011)), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

  18. Differentially expressed three non-coding alternate exons at 5' UTR of regulatory type I beta subunit gene of mouse.

    PubMed

    Banday, Abdul Rouf; Azim, Shafquat; Tabish, Mohammad

    2012-04-01

    Prkar1b gene encodes regulatory type I, beta subunit (RIβ) of cAMP dependent protein kinase A in mouse. Among the various isoforms of regulatory and catalytic subunits that comprise mammalian PKA, RIβ subunit is considered to be one of the important subunits for neuronal functions. This is involved in multiple forms of synaptic plasticity, and influences memory and learning by maintaining hippocampal long-term potentiation (LTP). Deficient expression of this gene has been implicated in autoimmune disease systemic lupus erythematosus (SLE). We have identified two novel non-coding exons of the Prkar1b gene (designated as exon 1A and exon 1B), which are spliced to the canonical exon 2 and constitute the 5' untranslated region giving rise to three alternative transcript isoforms. We have also confirmed the expression of the previously known first exon (designated as exon 1C) with known transcript published earlier. The transcripts containing exons 1A, 1B and 1C are differentially regulated during the development and tissue types. In silico study of more than 20 kb nucleotide sequence upstream of known translational initiation codon revealed three distinct promoter regions named as PA, PB, and PC upstream of the exon 1A, exon 1B and exon 1C respectively. PB is non-CpG related promoter but PA and PC are CpG related promoters, however all three promoters are TATA less. Further analysis showed that these promoters possess potential signature sequences for common as well as different transcription factors suggesting complex regulation of Prkar1b gene.

  19. Morphology and genetics reveal an intriguing pattern of differentiation at a very small geographic scale in a bird species, the forest thrush Turdus lherminieri

    PubMed Central

    Arnoux, E; Eraud, C; Navarro, N; Tougard, C; Thomas, A; Cavallo, F; Vetter, N; Faivre, B; Garnier, S

    2014-01-01

    Mobile organisms are expected to show population differentiation only over fairly large geographical distances. However, there is growing evidence of discrepancy between dispersal potential and realized gene flow. Here we report an intriguing pattern of differentiation at a very small spatial scale in the forest thrush (Turdus lherminieri), a bird species endemic to the Lesser Antilles. Analysis of 331 individuals from 17 sampling sites distributed over three islands revealed a clear morphological and genetic differentiation between these islands isolated by 40–50 km. More surprisingly, we found that the phenotypic divergence between the two geographic zones of the island of Guadeloupe was associated with a very strong genetic differentiation (Fst from 0.073–0.153), making this pattern a remarkable case in birds given the very small spatial scale considered. Molecular data (mitochondrial control region sequences and microsatellite genotypes) suggest that this strong differentiation could have occurred in situ, although alternative hypotheses cannot be fully discarded. This study suggests that the ongoing habitat fragmentation, especially in tropical forests, may have a deeper impact than previously thought on avian populations. PMID:24984605

  20. Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

    PubMed Central

    de Vries, Erik; Tscherne, Donna M.; Wienholts, Marleen J.; Cobos-Jiménez, Viviana; Scholte, Florine; García-Sastre, Adolfo; Rottier, Peter J. M.; de Haan, Cornelis A. M.

    2011-01-01

    Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis. PMID:21483486

  1. Genomic Profiling Reveals an Alternate Mechanism for Hepatic Tumor Promotion by Perfluorooctanoic Acid in Rainbow Trout

    PubMed Central

    Tilton, Susan C.; Orner, Gayle A.; Benninghoff, Abby D.; Carpenter, Hillary M.; Hendricks, Jerry D.; Pereira, Cliff B.; Williams, David E.

    2008-01-01

    Background Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are considered refractory to carcinogenesis by PPs. Previous studies with rainbow trout indicate they are also insensitive to peroxisome proliferation by the PP dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure. Objectives In this study, we used trout as a unique in vivo tumor model to study the potential for PFOA carcinogenesis in the absence of peroxisome proliferation compared with the structurally diverse PPs clofibrate (CLOF) and DHEA. Mechanisms of carcinogenesis were identified from hepatic gene expression profiles phenotypically anchored to tumor outcome. Methods We fed aflatoxin B1 or sham-initiated animals 200–1,800 ppm PFOA in the diet for 30 weeks for tumor analysis. We subsequently examined gene expression by cDNA array in animals fed PFOA, DHEA, CLOF, or 5 ppm 17β-estradiol (E2, a known tumor promoter) in the diet for 14 days. Results PFOA (1,800 ppm or 50 mg/kg/day) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity (p < 0.0001), whereas CLOF showed no effect. Carcinogenesis was independent of peroxisome proliferation, measured by lack of peroxisomal β-oxidation and catalase activity. Alternately, both tumor promoters, PFOA and DHEA, resulted in estrogenic gene signatures with strong correlation to E2 by Pearson correlation (R = 0.81 and 0.78, respectively), whereas CLOF regulated no genes in common with E2. Conclusions These data suggest that the tumor-promoting activities of PFOA in trout are due to novel mechanisms involving estrogenic signaling and are independent of peroxisome proliferation. PMID:18709148

  2. Functional photoreceptor loss revealed with adaptive optics: an alternate cause of color blindness.

    PubMed

    Carroll, Joseph; Neitz, Maureen; Hofer, Heidi; Neitz, Jay; Williams, David R

    2004-06-01

    There is enormous variation in the X-linked L/M (long/middle wavelength sensitive) gene array underlying "normal" color vision in humans. This variability has been shown to underlie individual variation in color matching behavior. Recently, red-green color blindness has also been shown to be associated with distinctly different genotypes. This has opened the possibility that there may be important phenotypic differences within classically defined groups of color blind individuals. Here, adaptive optics retinal imaging has revealed a mechanism for producing dichromatic color vision in which the expression of a mutant cone photopigment gene leads to the loss of the entire corresponding class of cone photoreceptor cells. Previously, the theory that common forms of inherited color blindness could be caused by the loss of photoreceptor cells had been discounted. We confirm that remarkably, this loss of one-third of the cones does not impair any aspect of vision other than color.

  3. ExactDAS: an exact test procedure for the detection of differential alternative splicing in microarray experiments.

    PubMed

    Mary-Huard, Tristan; Jaffrezic, Florence; Robin, Stéphane

    2012-11-06

    The aim of this paper is to propose a test procedure for the detection of differential alternative splicing across conditions for tiling array or exon chip data. While developed in a mixed model framework, the test procedure is exact (avoiding computational burden) and applicable to a large variety of contrasts, including several previously published ones. A simulation study is presented to evaluate the robustness and performance of the method. It is found to have a good detection power of genes under differential alternative splicing, even for five biological replicates and four probes per exon. The methodology also enables the comparison of various experimental designs through exact power curves. This is illustrated with the comparison of paired and unpaired experiments. The test procedure was applied to two publicly available cancer data sets based on exon arrays, and showed promising results.

  4. 'Living stones' reveal alternative petal identity programs within the core eudicots.

    PubMed

    Brockington, Samuel F; Rudall, Paula J; Frohlich, Michael W; Oppenheimer, David G; Soltis, Pamela S; Soltis, Douglas E

    2012-01-01

    Petals, defined as the showy laminar floral organs in the second floral whorl, have been shown to be under similar genetic control in distantly related core eudicot model organisms. On the basis of these findings, it is commonly assumed that the petal identity program regulated by B-class MADS-box gene homologs is invariant across the core eudicot clade. However, the core eudicots, which comprise >70% of angiosperm species, exhibit numerous instances of petal and sepal loss, transference of petal function between floral whorls, and recurrent petal evolution. In the face of these complex patterns of perianth evolution, the concept of a core eudicot petal identity program has not been tested. We therefore examined the petal identity program in the Caryophyllales, a core eudicot clade in which perianth differentiation into sepals and petals has evolved multiple times. Specifically, we analyzed the expression patterns of B- and C-class MADS-box homologs for evidence of a conserved petal identity program between sepal-derived and stamen-derived petaloid organs in the 'living stone' family Aizoaceae. We found that neither sepal-derived nor stamen-derived petaloid organs exhibit gene expression patterns consistent with the core eudicot petal identity program. B-class gene homologs are not expressed during the development of sepal-derived petals and are not implicated in petal identity in stamen-derived petals, as their transient expression coincides with early expression of the C-class homolog. We therefore provide evidence for petal development that is independent of B-class genes and suggest that different genetic control of petal identity has evolved within this lineage of core eudicots. These findings call for a more comprehensive understanding of perianth variation and its genetic causes within the core eudicots--an endeavor that will have broader implications for the interpretation of perianth evolution across angiosperms.

  5. Subtractive transcriptome analysis of leaf and rhizome reveals differentially expressed transcripts in Panax sokpayensis.

    PubMed

    Gurung, Bhusan; Bhardwaj, Pardeep K; Talukdar, Narayan C

    2016-11-01

    In the present study, suppression subtractive hybridization (SSH) strategy was used to identify rare and differentially expressed transcripts in leaf and rhizome tissues of Panax sokpayensis. Out of 1102 randomly picked clones, 513 and 374 high quality expressed sequenced tags (ESTs) were generated from leaf and rhizome subtractive libraries, respectively. Out of them, 64.92 % ESTs from leaf and 69.26 % ESTs from rhizome SSH libraries were assembled into different functional categories, while others were of unknown function. In particular, ESTs encoding galactinol synthase 2, ribosomal RNA processing Brix domain protein, and cell division cycle protein 20.1, which are involved in plant growth and development, were most abundant in the leaf SSH library. Other ESTs encoding protein KIAA0664 homologue, ubiquitin-activating enzyme e11, and major latex protein, which are involved in plant immunity and defense response, were most abundant in the rhizome SSH library. Subtractive ESTs also showed similarity with genes involved in ginsenoside biosynthetic pathway, namely farnesyl pyrophosphate synthase, squalene synthase, and dammarenediol synthase. Expression profiles of selected ESTs validated the quality of libraries and confirmed their differential expression in the leaf, stem, and rhizome tissues. In silico comparative analyses revealed that around 13.75 % of unigenes from the leaf SSH library were not represented in the available leaf transcriptome of Panax ginseng. Similarly, around 18.12, 23.75, 25, and 6.25 % of unigenes from the rhizome SSH library were not represented in available root/rhizome transcriptomes of P. ginseng, Panax notoginseng, Panax quinquefolius, and Panax vietnamensis, respectively, indicating a major fraction of novel ESTs. Therefore, these subtractive transcriptomes provide valuable resources for gene discovery in P. sokpayensis and would complement the available transcriptomes from other Panax species.

  6. Histone modification profiling reveals differential signatures associated with human embryonic stem cell self-renewal and differentiation

    PubMed Central

    Bhanu, Natarajan V.; Sidoli, Simone; Garcia, Benjamin A.

    2016-01-01

    In this study, we trace developmental stages using epigenome changes in human embryonic stem cells (hESCs) treated with drugs modulating either self-renewal or differentiation. Based on microscopy, qPCR and flow cytometry, we classified the treatment outcome as inducing pluripotency (hESC, flurbiprofen and gatifloxacin), mesendoderm (sinomenine), differentiation (cyamarin, digoxin, digitoxin, selegeline and theanine) and lineage-commitment (RA). When we analyzed histone PTMs that imprinted these gene and protein expressions, the above classification was reassorted. Hyperacetylation at H3K4, 9, 14, 18, 56 and 122 as well as H4K5, 8, 12 and 16 emerged as the pluripotency signature of hESCs. Methylations especially of H3 at K9, K20, K27 and K36 characterized differentiation initiation as seen in no-drug control and fluribiprofen. Sinomenine-treated cells clustered close to “differentiation initiators”, consistent with flow cytometry where it induced mesendoderm, along with cyamarin and possibly selegnine. Neurectoderm, induced by RA and theanine manifested methylations on H3 shifts to H3.3. By both flow cytometry and histone PTM clustering, it appears that cells treated with gatifloxacin, flurbiprofen, digitoxin and digoxin were not yet lineage-committed or mixed cell types. Taken together, our moderate-throughput histone PTM profiling approach highlighted subtle epigenetic signatures that permitted us to predict divergent lineage progression even in differentiating cells with similar phenotype and gene expression. PMID:26631989

  7. The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators

    PubMed Central

    Llorian, Miriam; Gooding, Clare; Bellora, Nicolas; Hallegger, Martina; Buckroyd, Adrian; Wang, Xiao; Rajgor, Dipen; Kayikci, Melis; Feltham, Jack; Ule, Jernej; Eyras, Eduardo; Smith, Christopher W.J.

    2016-01-01

    Alternative splicing (AS) is a key component of gene expression programs that drive cellular differentiation. Smooth muscle cells (SMCs) are important in the function of a number of physiological systems; however, investigation of SMC AS has been restricted to a handful of events. We profiled transcriptome changes in mouse de-differentiating SMCs and observed changes in hundreds of AS events. Exons included in differentiated cells were characterized by particularly weak splice sites and by upstream binding sites for Polypyrimidine Tract Binding protein (PTBP1). Consistent with this, knockdown experiments showed that that PTBP1 represses many smooth muscle specific exons. We also observed coordinated splicing changes predicted to downregulate the expression of core components of U1 and U2 snRNPs, splicing regulators and other post-transcriptional factors in differentiated cells. The levels of cognate proteins were lower or similar in differentiated compared to undifferentiated cells. However, levels of snRNAs did not follow the expression of splicing proteins, and in the case of U1 snRNP we saw reciprocal changes in the levels of U1 snRNA and U1 snRNP proteins. Our results suggest that the AS program in differentiated SMCs is orchestrated by the combined influence of auxiliary RNA binding proteins, such as PTBP1, along with altered activity and stoichiometry of the core splicing machinery. PMID:27317697

  8. Functional Cross-Talking between Differentially Expressed and Alternatively Spliced Genes in Human Liver Cancer Cells Treated with Berberine.

    PubMed

    Sheng, Zhen; Sun, Yi; Zhu, Ruixin; Jiao, Na; Tang, Kailin; Cao, Zhiwei; Ma, Chao

    2015-01-01

    Berberine has been identified with anti-proliferative effects on various cancer cells. Many researchers have been trying to elucidate the anti-cancer mechanisms of berberine based on differentially expressed genes. However, differentially alternative splicing genes induced by berberine might also contribute to its pharmacological actions and have not been reported yet. Moreover, the potential functional cross-talking between the two sets of genes deserves further exploration. In this study, RNA-seq technology was used to detect the differentially expressed genes and differentially alternative spliced genes in BEL-7402 cancer cells induced by berberine. Functional enrichment analysis indicated that these genes were mainly enriched in the p53 and cell cycle signalling pathway. In addition, it was statistically proven that the two sets of genes were locally co-enriched along chromosomes, closely connected to each other based on protein-protein interaction and functionally similar on Gene Ontology tree. These results suggested that the two sets of genes regulated by berberine might be functionally cross-talked and jointly contribute to its cell cycle arresting effect. It has provided new clues for further researches on the pharmacological mechanisms of berberine as well as the other botanical drugs.

  9. Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

    PubMed

    Yoshida, Masa-aki; Yamada, Lixy; Ochi, Hiroe; Iwata, Yoko; Tamura-Nakano, Miwa; Sawada, Hitoshi; Sauer, Warwick H H; Ogura, Atsushi; Hirohashi, Noritaka

    2014-08-01

    In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa.

  10. Multitaxon activity profiling reveals differential microbial response to reduced seawater pH and oil pollution.

    PubMed

    Coelho, Francisco J R C; Cleary, Daniel F R; Costa, Rodrigo; Ferreira, Marina; Polónia, Ana R M; Silva, Artur M S; Simões, Mário M Q; Oliveira, Vanessa; Gomes, Newton C M

    2016-09-01

    There is growing concern that predicted changes to global ocean chemistry will interact with anthropogenic pollution to significantly alter marine microbial composition and function. However, knowledge of the compounding effects of climate change stressors and anthropogenic pollution is limited. Here, we used 16S and 18S rRNA (cDNA)-based activity profiling to investigate the differential responses of selected microbial taxa to ocean acidification and oil hydrocarbon contamination under controlled laboratory conditions. Our results revealed that a lower relative abundance of sulphate-reducing bacteria (Desulfosarcina/Desulfococcus clade) due to an adverse effect of seawater acidification and oil hydrocarbon contamination (reduced pH-oil treatment) may be coupled to changes in sediment archaeal communities. In particular, we observed a pronounced compositional shift and marked reduction in the prevalence of otherwise abundant operational taxonomic units (OTUs) belonging to the archaeal Marine Benthic Group B and Marine Hydrothermal Vent Group (MHVG) in the reduced pH-oil treatment. Conversely, the abundance of several putative hydrocarbonoclastic fungal OTUs was higher in the reduced pH-oil treatment. Sediment hydrocarbon profiling, furthermore, revealed higher concentrations of several alkanes in the reduced pH-oil treatment, corroborating the functional implications of the structural changes to microbial community composition. Collectively, our results advance the understanding of the response of a complex microbial community to the interaction between reduced pH and anthropogenic pollution. In future acidified marine environments, oil hydrocarbon contamination may alter the typical mixotrophic and k-/r-strategist composition of surface sediment microbiomes towards a more heterotrophic state with lower doubling rates, thereby impairing the ability of the ecosystem to recover from acute oil contamination events.

  11. Differentially expressed genes and interacting pathways in bladder cancer revealed by bioinformatic analysis.

    PubMed

    Shen, Yinzhou; Wang, Xuelei; Jin, Yongchao; Lu, Jiasun; Qiu, Guangming; Wen, Xiaofei

    2014-10-01

    The goal of this study was to identify cancer-associated differentially expressed genes (DEGs), analyze their biological functions and investigate the mechanism(s) of cancer occurrence and development, which may provide a theoretical foundation for bladder cancer (BCa) therapy. We downloaded the mRNA expression profiling dataset GSE13507 from the Gene Expression Omnibus database; the dataset includes 165 BCa and 68 control samples. T‑tests were used to identify DEGs. To further study the biological functions of the identified DEGs, we performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Next, we built a network of potentially interacting pathways to study the synergistic relationships among DEGs. A total of 12,105 genes were identified as DEGs, of which 5,239 were upregulated and 6,866 were downregulated in BCa. The DEGs encoding activator protein 1 (AP-1), nuclear factor of activated T-cells (NFAT) proteins, nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and interleukin (IL)-10 were revealed to participate in the significantly enriched immune pathways that were downregulated in BCa. KEGG enrichment analysis revealed 7 significantly upregulated and 47 significantly downregulated pathways enriched among the DEGs. We found a crosstalk interaction among a total of 44 pathways in the network of BCa-affected pathways. In conclusion, our results show that BCa involves dysfunctions in multiple systems. Our study is expected to pave ways for immune and inflammatory research and provide molecular insights for cancer therapy.

  12. Brain slice invasion model reveals genes differentially regulated in glioma invasion

    SciTech Connect

    Holtkamp, Nikola . E-mail: nikola.holtkamp@charite.de; Afanasieva, Anastasia; Elstner, Anja; Landeghem, Frank K.H. van; Koenneker, Matthias; Kuhn, Susanne A.; Kettenmann, Helmut; Deimling, Andreas von

    2005-11-04

    Invasion of tumor cells into adjacent brain areas is one of the major problems in treatment of glioma patients. To identify genes that might contribute to invasion, fluorescent F98 glioma cells were allowed to invade an organotypic brain slice. Gene expression analysis revealed 5 up-regulated and 14 down-regulated genes in invasive glioma cells as compared to non-invasive glioma cells. Two gene products, ferritin and cyclin B1, were verified in human gliomas by immunohistochemistry. Ferritin exhibited high mRNA levels in migratory F98 cells and also showed higher protein expression in the infiltrating edge of human gliomas. Cyclin B1 with high mRNA expression levels in stationary F98 cells showed marked protein expression in the central portions of gliomas. These findings are compatible with the concept of tumor cells either proliferating or migrating. Our study is the first to apply brain slice cultures for the identification of differentially regulated genes in glioma invasion.

  13. Intestinal transcriptome analysis revealed differential salinity adaptation between two tilapiine species.

    PubMed

    Ronkin, Dana; Seroussi, Eyal; Nitzan, Tali; Doron-Faigenboim, Adi; Cnaani, Avner

    2015-03-01

    Tilapias are a group of freshwater species, which vary in their ability to adapt to high salinity water. Osmotic regulation in fish is conducted mainly in the gills, kidney, and gastrointestinal tract (GIT). The mechanisms involved in ion and water transport through the GIT is not well-characterized, with only a few described complexes. Comparing the transcriptome of the anterior and posterior intestinal sections of a freshwater and saltwater adapted fish by deep-sequencing, we examined the salinity adaptation of two tilapia species: the high salinity-tolerant Oreochromis mossambicus (Mozambique tilapia), and the less salinity-tolerant Oreochromis niloticus (Nile tilapia). This comparative analysis revealed high similarity in gene expression response to salinity change between species in the posterior intestine and large differences in the anterior intestine. Furthermore, in the anterior intestine 68 genes were saltwater up-regulated in one species and down-regulated in the other species (47 genes up-regulated in O. niloticus and down-regulated in O. mossambicus, with 21 genes showing the reverse pattern). Gene ontology (GO) analysis showed a high proportion of transporter and ion channel function among these genes. The results of this study point to a group of genes that differed in their salinity-dependent regulation pattern in the anterior intestine as potentially having a role in the differential salinity tolerance of these two closely related species.

  14. Proteomic Analysis of Fetal Ovaries Reveals That Primordial Follicle Formation and Transition Are Differentially Regulated

    PubMed Central

    Xu, Mengmeng; Che, Long; Yang, Zhenguo; Zhang, Pan; Shi, Jiankai; Li, Jian; Lin, Yan; Fang, Zhengfeng; Che, Lianqiang; Feng, Bin; Wu, De

    2017-01-01

    Primordial follicle formation represents a critical phase of the initiation of embryonic reproductive organ development, while the primordial follicle transition into primary follicle determines whether oestrus or ovulation will occur in female animals. To identify molecular mechanism of new proteins which are involved in ovarian development, we employed 2D-DIGE to compare the protein expression profiles of primordial follicles and primary follicles of fetal ovaries in pigs. Fetal ovaries were collected at distinct time-points of the gestation cycle (g55 and g90). The identified proteins at the g55 time-point are mainly involved in the development of anatomical structures [reticulocalbin-1 (RCN1), reticulocalbin-3 (RCN3)], cell differentiation (actin), and stress response [heterogeneous nuclear ribonucleoprotein K (HNRNPK)]. Meanwhile, at the g90 stage, the isolated proteins with altered expression levels were mainly associated with cell proliferation [major vault protein (MVP)] and stress response [heat shock-related 70 kDa protein 2 (HSPA2)]. In conclusion, our work revealed that primordial follicle formation is regulated by RCN1, RCN3, actin, and HNRNPK, while the primordial follicle transformation to primary follicle is regulated by MVP and HSPA2. Therefore, our results provide further information for the prospective understanding of the molecular mechanism(s) involved in the regulation of the ovarian follicle development. PMID:28265575

  15. Differential gene expression and alternative splicing between diploid and tetraploid watermelon lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Synthetic tetraploid plants have been used for production of seedless triploid watermelon lines being pollinated with diploid plants. When compared to their diploid or triploid counterparts, the tetraploid exhibit wide phenotypic differences. Though many factors, including alternative splicing (AS),...

  16. Effects of differential rates of alternative reinforcement on resurgence of human behavior.

    PubMed

    Smith, Brooke M; Smith, Gregory S; Shahan, Timothy A; Madden, Gregory J; Twohig, Michael P

    2017-01-01

    Despite the success of exposure-based psychotherapies in anxiety treatment, relapse remains problematic. Resurgence, the return of previously eliminated behavior following the elimination of an alternative source of reinforcement, is a promising model of operant relapse. Nonhuman resurgence research has shown that higher rates of alternative reinforcement result in faster, more comprehensive suppression of target behavior, but also in greater resurgence when alternative reinforcement is eliminated. This study investigated rich and lean rates of alternative reinforcement on response suppression and resurgence in typically developing humans. In Phase 1, three groups (Rich, n = 18; Lean, n = 18; Control, n = 10) acquired the target response. In Phase 2, target responding was extinguished and alternative reinforcement delivered on RI 1 s, RI 3 s, and extinction schedules, respectively. Resurgence was assessed during Phase 3 under extinction conditions for all groups. Target responding was suppressed most thoroughly in Rich and partially in Lean. Target responding resurged in the Rich and Lean groups, but not in the Control group. Between groups, resurgence was more pronounced in the Rich group than the Lean and Control groups. Clinical implications of these findings, including care on the part of clinicians when identifying alternative sources of reinforcement, are discussed.

  17. Differentiation of long-chain fatty acid oxidation disorders using alternative precursors and acylcarnitine profiling in fibroblasts.

    PubMed

    Roe, D S; Yang, B Z; Vianey-Saban, C; Struys, E; Sweetman, L; Roe, C R

    2006-01-01

    The differentiation of carnitine-acylcarnitine translocase deficiency (CACT) from carnitine palmitoyltransferase type II deficiency (CPT-II) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency from mitochondrial trifunctional protein deficiency (MTP) continues to be ambiguous using current acylcarnitine profiling techniques either from plasma or blood spots, or in the intact cell system (fibroblasts/amniocytes). Currently, enzyme assays are required to unequivocally differentiate CACT from CPT-II, and LCHAD from MTP. Over the years we have studied the responses of numerous FOD deficient cell lines to both even and odd numbered fatty acids of various chain lengths as well as branched-chain amino acids. In doing so, we discovered diagnostic elevations of unlabeled butyrylcarnitine detected only in CACT deficient cell lines when incubated with a shorter chain fatty acid, [7-2H3]heptanoate plus l-carnitine compared to the routinely used long-chain fatty acid, [16-2H3]palmitate. In monitoring the unlabeled C4/C5 acylcarnitine ratio, further differentiation from ETF/ETF-DH is also achieved. Similarly, incubating LCHAD and MTP deficient cell lines with the long-chain branched fatty acid, pristanic acid, and monitoring the C11/C9 acylcarnitine ratio has allowed differentiation between these disorders. These methods may be considered useful alternatives to specific enzyme assays for differentiation between these long-chain fatty acid oxidation disorders, as well as provide insight into new treatment strategies.

  18. Differential Expression of Alternative Oxidase Genes in Soybean Cotyledons during Postgerminative Development1

    PubMed Central

    McCabe, Tulene C.; Finnegan, Patrick M.; Harvey Millar, A.; Day, David A.; Whelan, James

    1998-01-01

    The expression of the alternative oxidase (AOX) was investigated during cotyledon development in soybean (Glycine max [L.] Merr.) seedlings. The total amount of AOX protein increased throughout development, not just in earlier stages as previously thought, and was correlated with the increase in capacity of the alternative pathway. Each AOX isoform (AOX1, AOX2, and AOX3) showed a different developmental trend in mRNA abundance, such that the increase in AOX protein and capacity appears to involve a shift in gene expression from AOX2 to AOX3. As the cotyledons aged, the size of the mitochondrial ubiquinone pool decreased. We discuss how this and other factors may affect the alternative pathway activity that results from the developmental regulation of AOX expression. PMID:9765553

  19. Comorbid Analysis of Genes Associated with Autism Spectrum Disorders Reveals Differential Evolutionary Constraints

    PubMed Central

    David, Maude M.; Enard, David; Ozturk, Alp; Daniels, Jena; Jung, Jae-Yoon; Diaz-Beltran, Leticia; Wall, Dennis. P.

    2016-01-01

    The burden of comorbidity in Autism Spectrum Disorder (ASD) is substantial. The symptoms of autism overlap with many other human conditions, reflecting common molecular pathologies suggesting that cross-disorder analysis will help prioritize autism gene candidates. Genes in the intersection between autism and related conditions may represent nonspecific indicators of dysregulation while genes unique to autism may play a more causal role. Thorough literature review allowed us to extract 125 ICD-9 codes comorbid to ASD that we mapped to 30 specific human disorders. In the present work, we performed an automated extraction of genes associated with ASD and its comorbid disorders, and found 1031 genes involved in ASD, among which 262 are involved in ASD only, with the remaining 779 involved in ASD and at least one comorbid disorder. A pathway analysis revealed 13 pathways not involved in any other comorbid disorders and therefore unique to ASD, all associated with basal cellular functions. These pathways differ from the pathways associated with both ASD and its comorbid conditions, with the latter being more specific to neural function. To determine whether the sequence of these genes have been subjected to differential evolutionary constraints, we studied long term constraints by looking into Genomic Evolutionary Rate Profiling, and showed that genes involved in several comorbid disorders seem to have undergone more purifying selection than the genes involved in ASD only. This result was corroborated by a higher dN/dS ratio for genes unique to ASD as compare to those that are shared between ASD and its comorbid disorders. Short-term evolutionary constraints showed the same trend as the pN/pS ratio indicates that genes unique to ASD were under significantly less evolutionary constraint than the genes associated with all other disorders. PMID:27414027

  20. Differential Molecular Responses of Rapeseed Cotyledons to Light and Dark Reveal Metabolic Adaptations toward Autotrophy Establishment

    PubMed Central

    He, Dongli; Damaris, Rebecca N.; Fu, Jinlei; Tu, Jinxing; Fu, Tingdong; Xi, Chen; Yi, Bin; Yang, Pingfang

    2016-01-01

    Photosynthesis competent autotrophy is established during the postgerminative stage of plant growth. Among the multiple factors, light plays a decisive role in the switch from heterotrophic to autotrophic growth. Under dark conditions, the rapeseed hypocotyl extends quickly with an apical hook, and the cotyledon is yellow and folded, and maintains high levels of the isocitrate lyase (ICL). By contrast, in the light, the hypocotyl extends slowly, the cotyledon unfolds and turns green, the ICL content changes in parallel with cotyledon greening. To reveal metabolic adaptations during the establishment of postgerminative autotrophy in rapeseed, we conducted comparative proteomic and metabolomic analyses of the cotyledons of seedlings grown under light versus dark conditions. Under both conditions, the increase in proteases, fatty acid β-oxidation and glyoxylate-cycle related proteins was accompanied by rapid degradation of the stored proteins and lipids with an accumulation of the amino acids. While light condition partially retarded these conversions. Light significantly induced the expression of chlorophyll-binding and photorespiration related proteins, resulting in an increase in reducing-sugars. However, the levels of some chlorophyllide conversion, Calvin-cycle and photorespiration related proteins also accumulated in dark grown cotyledons, implying that the transition from heterotrophy to autotrophy is programmed in the seed rather than induced by light. Various anti-stress systems, e.g., redox related proteins, salicylic acid, proline and chaperones, were employed to decrease oxidative stress, which was mainly derived from lipid oxidation or photorespiration, under both conditions. This study provides a comprehensive understanding of the differential molecular responses of rapeseed cotyledons to light and dark conditions, which will facilitate further study on the complex mechanism underlying the transition from heterotrophy to autotrophy. PMID:27471506

  1. Duplication and differentiation of common carp (Cyprinus carpio) myoglobin genes revealed by BAC analysis.

    PubMed

    Zhao, Zi-Xia; Xu, Peng; Cao, Ding-Chen; Kuang, You-Yi; Deng, Hai-Xia; Zhang, Yan; Xu, Li-Ming; Li, Jiong-Tang; Xu, Jian; Sun, Xiao-Wen

    2014-09-15

    Two distinct myoglobin (mb) transcripts have been reported in common carp, Cyprinus carpio, which is a hypoxia-tolerant fish living in habitats with greatly fluctuant dissolved oxygen levels. Recombinant protein analysis has shown functional specialization of the two mb transcripts. In this work, analysis for mb-containing bacterial artificial chromosome (BAC) clones indicated different genome loci for common carp myoglobin-1 (mb-1) and myoglobin-2 (mb-2) genes. Fluorescence in situ hybridization (FISH) revealed that mb-1 and mb-2 are located on separate chromosomes. In both of the mb-1 and mb-2 containing BAC clones, gene synteny was well conserved with the homologous region on zebrafish chromosome 1, supporting that the common carp specific mb-2 gene originated from the recent whole genome duplication event in cyprinid lineage. Transcription factor binding sites search indicated that both common carp mb genes lacked specificity Protein 1 (Sp1) and myocyte enhancer factor-2 (MEF2) binding sites, which mediated muscle-specific and calcium-dependent expression in the well-studied mb promoters. Potential hypoxia response elements (HREs) were predicted in the regulatory region of common carp mb genes. These characteristics of common carp mb gene regulatory region well interpreted the hypoxia-inducible, non-muscle expression pattern of mb-1. In the case of mb-2, a 10 bp insertion to the binding site of upstream stimulatory factor (USF), which was a co-factor of hypoxia inducible factor (HIF), might cause the non-response to hypoxia treatment of mb-2. The case of common carp mb gene duplication and subsequent differentiation in expression pattern and protein function provided an example for adaptive evolution toward aquatic hypoxia tolerance.

  2. A large-scale analysis of alternative splicing reveals a key role of QKI in lung cancer.

    PubMed

    de Miguel, Fernando J; Pajares, María J; Martínez-Terroba, Elena; Ajona, Daniel; Morales, Xabier; Sharma, Ravi D; Pardo, Francisco J; Rouzaut, Ana; Rubio, Angel; Montuenga, Luis M; Pio, Ruben

    2016-11-01

    Increasing interest has been devoted in recent years to the understanding of alternative splicing in cancer. In this study, we performed a genome-wide analysis to identify cancer-associated splice variants in non-small cell lung cancer. We discovered and validated novel differences in the splicing of genes known to be relevant to lung cancer biology, such as NFIB, ENAH or SPAG9. Gene enrichment analyses revealed an important contribution of alternative splicing to cancer-related molecular functions, especially those involved in cytoskeletal dynamics. Interestingly, a substantial fraction of the altered genes found in our analysis were targets of the protein quaking (QKI), pointing to this factor as one of the most relevant regulators of alternative splicing in non-small cell lung cancer. We also found that ESYT2, one of the QKI targets, is involved in cytoskeletal organization. ESYT2-short variant inhibition in lung cancer cells resulted in a cortical distribution of actin whereas inhibition of the long variant caused an increase of endocytosis, suggesting that the cancer-associated splicing pattern of ESYT2 has a profound impact in the biology of cancer cells. Finally, we show that low nuclear QKI expression in non-small cell lung cancer is an independent prognostic factor for disease-free survival (HR = 2.47; 95% CI = 1.11-5.46, P = 0.026). In conclusion, we identified several splicing variants with functional relevance in lung cancer largely regulated by the splicing factor QKI, a tumor suppressor associated with prognosis in lung cancer.

  3. The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

    PubMed

    Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W; Kulozik, Andreas E

    2016-09-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells.

  4. Modified Multiple-Choice Items for Alternate Assessments: Reliability, Difficulty, and Differential Boost

    ERIC Educational Resources Information Center

    Kettler, Ryan J.; Rodriguez, Michael C.; Bolt, Daniel M.; Elliott, Stephen N.; Beddow, Peter A.; Kurz, Alexander

    2011-01-01

    Federal policy on alternate assessment based on modified academic achievement standards (AA-MAS) inspired this research. Specifically, an experimental study was conducted to determine whether tests composed of modified items would have the same level of reliability as tests composed of original items, and whether these modified items helped reduce…

  5. Cost Differentials and the Treatment of Equipment Assets: An Analysis of Alternatives.

    ERIC Educational Resources Information Center

    Frohreich, Lloyd E.

    This paper is a discussion of alternative state approaches to aiding and costing capital outlay programs, particularly equipment purchases for vocational programs. Equipment costs for vocational programs tend to be a larger proportion of the total costs than in other programs. The paper includes a discussion of such topics as the magnitude of…

  6. Differential Coexpression Analysis Reveals Extensive Rewiring of Arabidopsis Gene Coexpression in Response to Pseudomonas syringae Infection

    PubMed Central

    Jiang, Zhenhong; Dong, Xiaobao; Li, Zhi-Gang; He, Fei; Zhang, Ziding

    2016-01-01

    Plant defense responses to pathogens involve massive transcriptional reprogramming. Recently, differential coexpression analysis has been developed to study the rewiring of gene networks through microarray data, which is becoming an important complement to traditional differential expression analysis. Using time-series microarray data of Arabidopsis thaliana infected with Pseudomonas syringae, we analyzed Arabidopsis defense responses to P. syringae through differential coexpression analysis. Overall, we found that differential coexpression was a common phenomenon of plant immunity. Genes that were frequently involved in differential coexpression tend to be related to plant immune responses. Importantly, many of those genes have similar average expression levels between normal plant growth and pathogen infection but have different coexpression partners. By integrating the Arabidopsis regulatory network into our analysis, we identified several transcription factors that may be regulators of differential coexpression during plant immune responses. We also observed extensive differential coexpression between genes within the same metabolic pathways. Several metabolic pathways, such as photosynthesis light reactions, exhibited significant changes in expression correlation between normal growth and pathogen infection. Taken together, differential coexpression analysis provides a new strategy for analyzing transcriptional data related to plant defense responses and new insights into the understanding of plant-pathogen interactions. PMID:27721457

  7. Transcriptional analysis of histone deacetylase family members reveal similarities between differentiating and aging spermatogonial stem cells.

    PubMed

    Kofman, Amber E; Huszar, Jessica M; Payne, Christopher J

    2013-02-01

    The differentiation of adult stem cells involves extensive chromatin remodeling, mediated in part by the gene products of histone deacetylase (HDAC) family members. While the transcriptional downregulation of HDACs can impede stem cell self-renewal in certain contexts, it may also promote stem cell maintenance under other circumstances. In self-renewing, differentiating, and aging spermatogonial stem cells (SSCs), the gene expression dynamics of HDACs have not yet been characterized. To gain further insight with these studies, we analyzed the transcriptional profiles of six HDAC family members, previously identified to be the most highly expressed in self-renewing SSCs, during stem cell differentiation and aging. Here we discovered that in both differentiating and aging SSCs the expression of Sirt4 increases, while the expression of Hdac2, Hdac6, and Sirt1 decreases. When SSCs are exposed to the lifespan-enhancing drug rapamycin in vivo, the resultant HDAC gene expression patterns are opposite of those seen in the differentiating and aging SSCs, with increased Hdac2, Hdac6, and Sirt1 and decreased Hdac8, Hdac9, and Sirt4. Our findings suggest that HDACs important for stem cell maintenance and oxidative capacity are downregulated as adult stem cells differentiate or age. These results provide important insights into the epigenetic regulation of stem cell differentiation and aging in mammals.

  8. Geologically Controlled Isotope-Time Patterns Reveal Early Differentiation and Crust Formation Processes

    NASA Astrophysics Data System (ADS)

    Bennett, V. C.; Nutman, A. P.

    2014-12-01

    The mechanisms of continental crust production and evolution in the early Earth remain controversial, as are questions of the relative roles of early differentiation versus subsequent tectonic procssing in creating Earth's chemical signatures. Here we present geologic observations integrated with whole rock major, trace element and Sm-Nd isotopic signatures and combined with U-Pb and Lu-Hf isotopic compositions of zircon populations from the same rocks, from the most extensive early rock record comprising the 3.9 Ga to 3.6 Ga terranes of southwest Greenland. These data reveal repeated patterns of formation of juvenile TTG crust and associated mafic and ultramafic rocks in convergent margin settings followed by formation of more evolved granites [1]. Our new zircon Lu-Hf data from rare 3.6-3.7 Ga tonalites within the Itsaq Gneiss Complex, obtained from single component, non-migmatitic gneisses with simple zircon populations, limited within sample Hf isotopic variability and accurate U-Pb ages, now document extraction of juvenile tonalites from a near chondritic mantle source between 3.9 Ga and 3.6 Ga. The more evolved, granitic rocks in each area show slightly negative initial ɛHf in accord with crustal reworking of the older (3.8-3.9 Ga) gniesses. There is no evidence for Hadean material in the sources of the granitoids. The Hf isotope-time patterns are consistent with juvenile crust production from a mantle source that experienced only modest amounts of prior crustal extraction. They are distinct from those predicted by reprocessing of an enriched Hadean mafic crust, as has been proposed for this region [2] and for the source of the Hadean Jack Hills zircons [3]. The well-documented, time decreasing, positive 142Nd anomalies [e.g., 4] from these rocks are further evidence of crustal derivation from a convecting mantle source, rather than reworking of an enriched mafic lithosphere. The 143Nd isotopic -time patterns are more complex, reflecting the interplay

  9. Alternative NF-κB Regulates RANKL-induced Osteoclast Differentiation and Mitochondrial Biogenesis via Independent Mechanisms

    PubMed Central

    Zeng, Rong; Faccio, Roberta; Novack, Deborah V

    2016-01-01

    Mitochondrial biogenesis, the generation of new mitochondrial DNA and proteins, has been linked to osteoclast (OC) differentiation and function. In this study we used mice with mutations in key alternative NF-κB pathway proteins, RelB and NIK, to dissect the complex relationship between mitochondrial biogenesis and osteoclastogenesis. OC precursors lacking either NIK or RelB, RANKL were unable to increase mitochondrial DNA or OxPhos protein expression, associated with lower oxygen consumption rates. Transgenic OC precursors expressing constitutively active NIK showed normal RANKL-induced mitochondrial biogenesis (OxPhos expression and mitochondria copy number) compared to controls, but larger mitochondrial dimensions and increased oxygen consumption rates, suggesting increased mitochondrial function. To deduce the mechanism for mitochondrial biogenesis defects in NIK- and RelB-deficient precursors, we examined expression of genes known to control this process. PGC-1β (Ppargc1b) expression, but not PGC-1α, PPRC1 or ERRα, was significantly reduced in RelB−/− and NIK−/− OCs. Because PGC-1β has been reported to positively regulate both mitochondrial biogenesis and differentiation in OCs, we retrovirally overexpressed PGC-1β in RelB−/− cells, but surprisingly found that it did not affect differentiation, nor restore RANKL-induced mitochondrial biogenesis. To determine whether the blockade in osteoclastogenesis in RelB-deficient cells precludes mitochondrial biogenesis, we rescued RelB−/− differentiation via overexpression of NFATc1. Mitochondrial parameters in neither WT nor RelB-deficient cultures were affected by NFATc1 overexpression, and bone resorption in RelB −/− was not restored. Furthermore, NFATc1 co-overexpression with PGC-1β, while allowing OC differentiation, did not rescue mitochondrial biogenesis or bone resorption in RelB−/− OCs, by CTX-I levels. Thus, our results indicate that the alternative NF-κB pathway plays dual, but

  10. Genome-wide transcriptional profiling reveals molecular signatures of secondary xylem differentiation in Populus tomentosa.

    PubMed

    Yang, X H; Li, X G; Li, B L; Zhang, D Q

    2014-11-11

    Wood formation occurs via cell division, primary cell wall and secondary wall formation, and programmed cell death in the vascular cambium. Transcriptional profiling of secondary xylem differentiation is essential for understanding the molecular mechanisms underlying wood formation. Differential gene expression in secondary xylem differentiation of Populus has been previously investigated using cDNA microarray analysis. However, little is known about the molecular mechanisms from a genome-wide perspective. In this study, the Affymetrix poplar genome chips containing 61,413 probes were used to investigate the changes in the transcriptome during secondary xylem differentiation in Chinese white poplar (Populus tomentosa). Two xylem tissues (newly formed and lignified) were sampled for genome-wide transcriptional profiling. In total, 6843 genes (~11%) were identified with differential expression in the two xylem tissues. Many genes involved in cell division, primary wall modification, and cellulose synthesis were preferentially expressed in the newly formed xylem. In contrast, many genes, including 4-coumarate:cinnamate-4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), and caffeoyl CoA 3-O-methyltransferase (CCoAOMT), associated with lignin biosynthesis were more transcribed in the lignified xylem. The two xylem tissues also showed differential expression of genes related to various hormones; thus, the secondary xylem differentiation could be regulated by hormone signaling. Furthermore, many transcription factor genes were preferentially expressed in the lignified xylem, suggesting that wood lignification involves extensive transcription regulation. The genome-wide transcriptional profiling of secondary xylem differentiation could provide additional insights into the molecular basis of wood formation in poplar species.

  11. Single-nucleus RNA-seq of differentiating human myoblasts reveals the extent of fate heterogeneity

    PubMed Central

    Zeng, Weihua; Jiang, Shan; Kong, Xiangduo; El-Ali, Nicole; Ball, Alexander R.; Ma, Christopher I-Hsing; Hashimoto, Naohiro; Yokomori, Kyoko; Mortazavi, Ali

    2016-01-01

    Myoblasts are precursor skeletal muscle cells that differentiate into fused, multinucleated myotubes. Current single-cell microfluidic methods are not optimized for capturing very large, multinucleated cells such as myotubes. To circumvent the problem, we performed single-nucleus transcriptome analysis. Using immortalized human myoblasts, we performed RNA-seq analysis of single cells (scRNA-seq) and single nuclei (snRNA-seq) and found them comparable, with a distinct enrichment for long non-coding RNAs (lncRNAs) in snRNA-seq. We then compared snRNA-seq of myoblasts before and after differentiation. We observed the presence of mononucleated cells (MNCs) that remained unfused and analyzed separately from multi-nucleated myotubes. We found that while the transcriptome profiles of myoblast and myotube nuclei are relatively homogeneous, MNC nuclei exhibited significant heterogeneity, with the majority of them adopting a distinct mesenchymal state. Primary transcripts for microRNAs (miRNAs) that participate in skeletal muscle differentiation were among the most differentially expressed lncRNAs, which we validated using NanoString. Our study demonstrates that snRNA-seq provides reliable transcriptome quantification for cells that are otherwise not amenable to current single-cell platforms. Our results further indicate that snRNA-seq has unique advantage in capturing nucleus-enriched lncRNAs and miRNA precursors that are useful in mapping and monitoring differential miRNA expression during cellular differentiation. PMID:27566152

  12. Single-nucleus RNA-seq of differentiating human myoblasts reveals the extent of fate heterogeneity.

    PubMed

    Zeng, Weihua; Jiang, Shan; Kong, Xiangduo; El-Ali, Nicole; Ball, Alexander R; Ma, Christopher I-Hsing; Hashimoto, Naohiro; Yokomori, Kyoko; Mortazavi, Ali

    2016-12-01

    Myoblasts are precursor skeletal muscle cells that differentiate into fused, multinucleated myotubes. Current single-cell microfluidic methods are not optimized for capturing very large, multinucleated cells such as myotubes. To circumvent the problem, we performed single-nucleus transcriptome analysis. Using immortalized human myoblasts, we performed RNA-seq analysis of single cells (scRNA-seq) and single nuclei (snRNA-seq) and found them comparable, with a distinct enrichment for long non-coding RNAs (lncRNAs) in snRNA-seq. We then compared snRNA-seq of myoblasts before and after differentiation. We observed the presence of mononucleated cells (MNCs) that remained unfused and analyzed separately from multi-nucleated myotubes. We found that while the transcriptome profiles of myoblast and myotube nuclei are relatively homogeneous, MNC nuclei exhibited significant heterogeneity, with the majority of them adopting a distinct mesenchymal state. Primary transcripts for microRNAs (miRNAs) that participate in skeletal muscle differentiation were among the most differentially expressed lncRNAs, which we validated using NanoString. Our study demonstrates that snRNA-seq provides reliable transcriptome quantification for cells that are otherwise not amenable to current single-cell platforms. Our results further indicate that snRNA-seq has unique advantage in capturing nucleus-enriched lncRNAs and miRNA precursors that are useful in mapping and monitoring differential miRNA expression during cellular differentiation.

  13. Analysis of E-box DNA binding during myeloid differentiation reveals complexes that contain Mad but not Max.

    PubMed Central

    Ryan, K M; Birnie, G D

    1997-01-01

    It has been shown that during myeloid differentiation the levels of mad1 mRNA are induced before the loss of c-Myc protein. This suggests that inactivation of the differentiation-blocking activity of c-Myc might not occur primarily through the loss of Myc protein, but through an increase in the levels of its antagonist, Mad1. To investigate this question we have analysed the levels of mad1 mRNA during differentiation of myeloid leukaemic HL60 cells. Although levels of mad1 mRNA were moderately increased after induction with phorbol ester, we also found that differentiation could be achieved with other inducers without any concomitant up-regulation of mad1 mRNA. In addition, analysis of E-box DNA binding revealed that, although Myc-Max complexes were lost rapidly after differentiation induction, formation of Mad1-containing complexes only occurred during the later stages of the differentiation programme. Further analysis of these Mad-containing complexes revealed that they were also unlikely to have the capacity to antagonize c-Myc function, as they did not contain Max. Therefore these data suggest that an increase in the levels of mad1 mRNA or the formation of a Mad-Max complex are unlikely to be essential or determining events for myeloid differentiation. In addition, the discovery of DNA-binding complexes that contain Mad1, but not Max, opens up this transcription factor network to include other Max-like proteins or proteins of an unrelated nature. PMID:9224632

  14. [Differentiation of chum salmon Oncorhynchus keta Walbaum populations as revealed with microsatellite and allozyme markers: a comparison].

    PubMed

    Rubtsoba, G A; Afanas'ev, K I; Malinina, T V; Shitova, M V; Rakitskaia, T A; Prokhorovskaia, V D; Zhivotovskiĭ, L A

    2008-07-01

    The character and extent of population differentiation in chum salmon Oncorhynchus keta from Sakhalin and Iturup were comparatively studied with 10 microsatellite and 12 allozyme markers. It was demonstrated with the example of allozyme polymorphism at the EstD locus that the effect of an individual locus with one major allele is capable of distorting the total picture of population differentiation. Multiallelic microsatellites were more efficient in revealing the genetic structure of chum salmon populations at the levels of differences between regional populations and between the stocks of individual rivers of the same region.

  15. Boolean Modeling Reveals the Necessity of Transcriptional Regulation for Bistability in PC12 Cell Differentiation

    PubMed Central

    Offermann, Barbara; Knauer, Steffen; Singh, Amit; Fernández-Cachón, María L.; Klose, Martin; Kowar, Silke; Busch, Hauke; Boerries, Melanie

    2016-01-01

    The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation. PMID:27148350

  16. Boolean Modeling Reveals the Necessity of Transcriptional Regulation for Bistability in PC12 Cell Differentiation.

    PubMed

    Offermann, Barbara; Knauer, Steffen; Singh, Amit; Fernández-Cachón, María L; Klose, Martin; Kowar, Silke; Busch, Hauke; Boerries, Melanie

    2016-01-01

    The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation.

  17. Differential reinforcement of alternative behavior increases resistance to extinction: clinical demonstration, animal modeling, and clinical test of one solution.

    PubMed

    Mace, F Charles; McComas, Jennifer J; Mauro, Benjamin C; Progar, Patrick R; Taylor, Bridget; Ervin, Ruth; Zangrillo, Amanda N

    2010-05-01

    Basic research with pigeons on behavioral momentum suggests that differential reinforcement of alternative behavior (DRA) can increase the resistance of target behavior to change. This finding suggests that clinical applications of DRA may inadvertently increase the persistence of target behavior even as it decreases its frequency. We conducted three coordinated experiments to test whether DRA has persistence-strengthening effects on clinically significant target behavior and then tested the effectiveness of a possible solution to this problem in both a nonhuman and clinical study. Experiment 1 compared resistance to extinction following baseline rates of reinforcement versus higher DRA rates of reinforcement in a clinical study. Resistance to extinction was substantially greater following DRA. Experiment 2 tested a rat model of a possible solution to this problem. Training an alternative response in a context without reinforcement of the target response circumvented the persistence-strengthening effects of DRA. Experiment 3 translated the rat model into a novel clinical application of DRA. Training an alternative response with DRA in a separate context resulted in lower resistance to extinction than employing DRA in the context correlated with reinforcement of target behavior. The value of coordinated bidirectional translational research is discussed.

  18. Dental Pulp Stem Cells Differentiation Reveals New Insights in Oct4A Dynamics

    PubMed Central

    D'Aurizio, Federica; Puppato, Elisa; Pandolfi, Maura; Beltrami, Antonio Paolo; Cesselli, Daniela; Falini, Giuseppe; Beltrami, Carlo Alberto; Curcio, Francesco

    2012-01-01

    Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES) pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC). A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A). Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research. PMID:22844522

  19. Alternating Current Electric Fields of Varying Frequencies: Effects on Proliferation and Differentiation of Porcine Neural Progenitor Cells

    PubMed Central

    Lim, Ji-Hey; McCullen, Seth D.; Piedrahita, Jorge A.

    2013-01-01

    Abstract Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes. PMID:23961767

  20. Alternating current electric fields of varying frequencies: effects on proliferation and differentiation of porcine neural progenitor cells.

    PubMed

    Lim, Ji-Hey; McCullen, Seth D; Piedrahita, Jorge A; Loboa, Elizabeth G; Olby, Natasha J

    2013-10-01

    Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes.

  1. Integrative Multi-omic Analysis of Human Platelet eQTLs Reveals Alternative Start Site in Mitofusin 2

    PubMed Central

    Simon, Lukas M.; Chen, Edward S.; Edelstein, Leonard C.; Kong, Xianguo; Bhatlekar, Seema; Rigoutsos, Isidore; Bray, Paul F.; Shaw, Chad A.

    2016-01-01

    Platelets play a central role in ischemic cardiovascular events. Cardiovascular disease (CVD) is a major cause of death worldwide. Numerous genome-wide association studies (GWASs) have identified loci associated with CVD risk. However, our understanding of how these variants contribute to disease is limited. Using data from the platelet RNA and expression 1 (PRAX1) study, we analyzed cis expression quantitative trait loci (eQTLs) in platelets from 154 normal human subjects. We confirmed these results in silico by performing allele-specific expression (ASE) analysis, which demonstrated that the allelic directionality of eQTLs and ASE patterns correlate significantly. Comparison of platelet eQTLs with data from the Genotype-Tissue Expression (GTEx) project revealed that a number of platelet eQTLs are platelet specific and that platelet eQTL peaks localize to the gene body at a higher rate than eQTLs from other tissues. Upon integration with data from previously published GWASs, we found that the trait-associated variant rs1474868 coincides with the eQTL peak for mitofusin 2 (MFN2). Additional experimental and computational analyses revealed that this eQTL is linked to an unannotated alternate MFN2 start site preferentially expressed in platelets. Integration of phenotype data from the PRAX1 study showed that MFN2 expression levels were significantly associated with platelet count. This study links the variant rs1474868 to a platelet-specific regulatory role for MFN2 and demonstrates the utility of integrating multi-omic data with eQTL analysis in disease-relevant tissues for interpreting GWAS results. PMID:27132591

  2. Computational modeling reveals that a combination of chemotaxis and differential adhesion leads to robust cell sorting during tissue patterning.

    PubMed

    Tan, Rui Zhen; Chiam, Keng-Hwee

    2014-01-01

    Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.

  3. By Ounce or By Calorie: The Differential Effects of Alternative Sugar-Sweetened Beverage Tax Strategies

    PubMed Central

    Zhen, Chen; Brissette, Ian F.; Ruff, Ryan R.

    2014-01-01

    The obesity epidemic and excessive consumption of sugar-sweetened beverages have led to proposals of economics-based interventions to promote healthy eating in the United States. Targeted food and beverage taxes and subsidies are prominent examples of such potential intervention strategies. This paper examines the differential effects of taxing sugar-sweetened beverages by calories and by ounces on beverage demand. To properly measure the extent of substitution and complementarity between beverage products, we developed a fully modified distance metric model of differentiated product demand that endogenizes the cross-price effects. We illustrated the proposed methodology in a linear approximate almost ideal demand system, although other flexible demand systems can also be used. In the empirical application using supermarket scanner data, the product-level demand model consists of 178 beverage products with combined market share of over 90%. The novel demand model outperformed the conventional distance metric model in non-nested model comparison tests and in terms of the economic significance of model predictions. In the fully modified model, a calorie-based beverage tax was estimated to cost $1.40 less in compensating variation than an ounce-based tax per 3,500 beverage calories reduced. This difference in welfare cost estimates between two tax strategies is more than three times as much as the difference estimated by the conventional distance metric model. If applied to products purchased from all sources, a 0.04-cent per kcal tax on sugar-sweetened beverages is predicted to reduce annual per capita beverage intake by 5,800 kcal. PMID:25414517

  4. Whole-genome analysis revealed the positively selected genes during the differentiation of indica and temperate japonica rice.

    PubMed

    Sun, Xinli; Jia, Qi; Guo, Yuchun; Zheng, Xiujuan; Liang, Kangjing

    2015-01-01

    To investigate the selective pressures acting on the protein-coding genes during the differentiation of indica and japonica, all of the possible orthologous genes between the Nipponbare and 93-11 genomes were identified and compared with each other. Among these genes, 8,530 pairs had identical sequences, and 27,384 pairs shared more than 90% sequence identity. Only 2,678 pairs of genes displaying a Ka/Ks ratio significantly greater than one were revealed, and most of these genes contained only nonsynonymous sites. The genes without synonymous site were further analyzed with the SNP data of 1529 O. sativa and O. rufipogon accessions, and 1068 genes were identified to be under positive selection during the differentiation of indica and temperate japonica. The positively selected genes (PSGs) are unevenly distributed on 12 chromosomes, and the proteins encoded by the PSGs are dominant with binding, transferase and hydrolase activities, and especially enriched in the plant responses to stimuli, biological regulations, and transport processes. Meanwhile, the most PSGs of the known function and/or expression were involved in the regulation of biotic/abiotic stresses. The evidence of pervasive positive selection suggested that many factors drove the differentiation of indica and japonica, which has already started in wild rice but is much lower than in cultivated rice. Lower differentiation and less PSGs revealed between the Or-It and Or-IIIt wild rice groups implied that artificial selection provides greater contribution on the differentiation than natural selection. In addition, the phylogenetic tree constructed with positively selected sites showed that the japonica varieties exhibited more diversity than indica on differentiation, and Or-III of O. rufipogon exhibited more than Or-I.

  5. DNA Methylation Profiling Reveals Correlation of Differential Methylation Patterns with Gene Expression in Human Epilepsy.

    PubMed

    Wang, Liang; Fu, Xinwei; Peng, Xi; Xiao, Zheng; Li, Zhonggui; Chen, Guojun; Wang, Xuefeng

    2016-05-01

    DNA methylation plays important roles in regulating gene expression and has been reported to be related with epilepsy. This study aimed to define differential DNA methylation patterns in drug-refractory epilepsy patients and to investigate the role of DNA methylation in human epilepsy. We performed DNA methylation profiling in brain tissues from epileptic and control patients via methylated-cytosine DNA immunoprecipitation microarray chip. Differentially methylated loci were validated by bisulfite sequencing PCR, and the messenger RNA (mRNA) levels of candidate genes were evaluated by reverse transcriptase PCR. We found 224 genes that showed differential DNA methylation between epileptic patients and controls. Among the seven candidate genes, three genes (TUBB2B, ATPGD1, and HTR6) showed relative transcriptional regulation by DNA methylation. TUBB2B and ATPGD1 exhibited hypermethylation and decreased mRNA levels, whereas HTR6 displayed hypomethylation and increased mRNA levels in the epileptic samples. Our findings suggest that certain genes become differentially regulated by DNA methylation in human epilepsy.

  6. Proteomics identification of differentially expressed proteins associated with pollen germination and tube growth reveals characteristics of germinated Oryza sativa pollen.

    PubMed

    Dai, Shaojun; Chen, Taotao; Chong, Kang; Xue, Yongbiao; Liu, Siqi; Wang, Tai

    2007-02-01

    Mature pollen from most plant species is metabolically quiescent; however, after pollination, it germinates quickly and gives rise to a pollen tube to transport sperms into the embryo sac. Because methods for collecting a large amount of in vitro germinated pollen grains for transcriptomics and proteomics studies from model plants of Arabidopsis and rice are not available, molecular information about the germination developmental process is lacking. Here we describe a method for obtaining a large quantity of in vitro germinating rice pollen for proteomics study. Two-dimensional electrophoresis of approximately 2300 protein spots revealed 186 that were differentially expressed in mature and germinated pollen. Most showed a changed level of expression, and only 66 appeared to be specific to developmental stages. Furthermore 160 differentially expressed protein spots were identified on mass spectrometry to match 120 diverse protein species. These proteins involve different cellular and metabolic processes with obvious functional skew toward wall metabolism, protein synthesis and degradation, cytoskeleton dynamics, and carbohydrate/energy metabolism. Wall metabolism-related proteins are prominently featured in the differentially expressed proteins and the pollen proteome as compared with rice sporophytic proteomes. Our study also revealed multiple isoforms and differential expression patterns between isoforms of a protein. These results provide novel insights into pollen function specialization.

  7. Whole Genome Analyses of a Well-Differentiated Liposarcoma Reveals Novel SYT1 and DDR2 Rearrangements

    PubMed Central

    Egan, Jan B.; Barrett, Michael T.; Champion, Mia D.; Middha, Sumit; Lenkiewicz, Elizabeth; Evers, Lisa; Francis, Princy; Schmidt, Jessica; Shi, Chang-Xin; Van Wier, Scott; Badar, Sandra; Ahmann, Gregory; Kortuem, K. Martin; Boczek, Nicole J.; Fonseca, Rafael; Craig, David W.; Carpten, John D.; Borad, Mitesh J.; Stewart, A. Keith

    2014-01-01

    Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2. PMID:24505276

  8. Whole genome analyses of a well-differentiated liposarcoma reveals novel SYT1 and DDR2 rearrangements.

    PubMed

    Egan, Jan B; Barrett, Michael T; Champion, Mia D; Middha, Sumit; Lenkiewicz, Elizabeth; Evers, Lisa; Francis, Princy; Schmidt, Jessica; Shi, Chang-Xin; Van Wier, Scott; Badar, Sandra; Ahmann, Gregory; Kortuem, K Martin; Boczek, Nicole J; Fonseca, Rafael; Craig, David W; Carpten, John D; Borad, Mitesh J; Stewart, A Keith

    2014-01-01

    Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2.

  9. Differential gene expression and subcellular targeting of Arabidopsis glutathione S-transferase F8 is achieved through alternative transcription start sites.

    PubMed

    Thatcher, Louise F; Carrie, Chris; Andersson, Carol R; Sivasithamparam, Krishnapillai; Whelan, James; Singh, Karam B

    2007-09-28

    Glutathione S-transferases (GSTs) play major roles in the protection of plants from biotic and abiotic stresses through the detoxification of xenobiotics and toxic endogenous products. This report describes additional complexity in the regulation of the well characterized stress-responsive Arabidopsis thaliana GSTF8 promoter. This complexity results from the use of multiple transcription start sites (TSS) to give rise to alternate GSTF8 transcripts with the potential to produce two in-frame proteins differing only in their N-terminal sequence. In addition to the originally mapped TSS (Chen, W., Chao, G., and Singh, K. B. (1996) Plant J. 10, 955-966), a further nine TSS have been identified, with the majority clustered into a distinct group. The most 3' TSS gives rise to the major message (GSTF8-S) and the shorter form of the protein, whereas those originating from upstream TSS (GSTF8-L) are more weakly expressed and encode for the larger form of the protein. Differential tissue-specific and stress-responsive expression patterns were observed (e.g. GSTF8-L is more highly expressed in leaves compared with roots, whereas GSTF8-S expression has the opposite pattern and is much more stress-responsive). Analysis of GSTF8-L and GSTF8-S proteins demonstrated that GSTF8-L is solely targeted to plastids, whereas GSTF8-S is cytoplasmic. In silico analysis revealed potential conservation of GSTF8-S across a wide range of plants; in contrast, conservation of GSTF8-L was confined to the Brassicaceae. These studies demonstrate that alternate TSS of the GSTF8 promoter are used to confer differential tissue-specific and stress-responsive expression patterns as well as to target the same protein to two different subcellular localizations.

  10. Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD

    PubMed Central

    Joya, Xavier; Garcia-Algar, Oscar; Vall, Oriol; Pujades, Cristina

    2014-01-01

    Background The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development. PMID:25383948

  11. Two-dimensional correlation spectroscopy reveals coupled immunoglobulin regions of differential flexibility that influence stability.

    PubMed

    Kamerzell, Tim J; Middaugh, C Russell

    2007-08-28

    Despite the well-accepted importance of protein flexibility and dynamics in molecular recognition and conformational stability, our understanding of these relationships is incomplete. Immunoglobulin flexibility is essential for antigen binding and adaptation to diverse molecular shapes and sizes. The inherent flexibility of immunoglobulins also renders these molecules suitable for investigating the possible relationships between protein flexibility and stability. To better understand these inter-relationships, we employ generalized perturbation-based two-dimensional correlation FTIR spectroscopy to monitor the time evolution of H-D exchange of an IgG1 as a function of pH. The differential flexibility of various immunoglobulin regions is described in response to an external perturbation and shown to vary widely. The greatest number of regions with differential exchange rates and, thus differential flexibility, is seen at pH 6. Approximately seven, six, five, and four separate states that exchange with different rates were observed at pH 6, 8, 4, and 2, respectively. The overall distribution of exchange rates calculated from the decays of the integrated Amide I and Amide II areas provides further evidence of multiple regions with differential flexibility. The sequence of events at pH 4 determined from the asynchronous vibrational patterns is of significant interest and suggests protonation of Glu and Asp side chains occurs first and initiates changes in the conformation and flexibility of different sheet and turns structure. A complex inter-relationship between differential regional flexibility and conformational coupling (i.e., cooperativity) initiated by changes in pH influences the stability of this IgG.

  12. Transcriptomic Analysis of Thermally Stressed Symbiodinium Reveals Differential Expression of Stress and Metabolism Genes.

    PubMed

    Gierz, Sarah L; Forêt, Sylvain; Leggat, William

    2017-01-01

    Endosymbioses between dinoflagellate algae (Symbiodinium sp.) and scleractinian coral species form the foundation of coral reef ecosystems. The coral symbiosis is highly susceptible to elevated temperatures, resulting in coral bleaching, where the algal symbiont is released from host cells. This experiment aimed to determine the transcriptional changes in cultured Symbiodinium, to better understand the response of cellular mechanisms under future temperature conditions. Cultures were exposed to elevated temperatures (average 31°C) or control conditions (24.5°C) for a period of 28 days. Whole transcriptome sequencing of Symbiodinium cells on days 4, 19, and 28 were used to identify differentially expressed genes under thermal stress. A large number of genes representing 37.01% of the transcriptome (∼23,654 unique genes, FDR < 0.05) with differential expression were detected at no less than one of the time points. Consistent with previous studies of Symbiodinium gene expression, fold changes across the transcriptome were low, with 92.49% differentially expressed genes at ≤2-fold change. The transcriptional response included differential expression of genes encoding stress response components such as the antioxidant network and molecular chaperones, cellular components such as core photosynthesis machinery, integral light-harvesting protein complexes and enzymes such as fatty acid desaturases. Differential expression of genes encoding glyoxylate cycle enzymes were also found, representing the first report of this in Symbiodinium. As photosynthate transfer from Symbiodinium to coral hosts provides up to 90% of a coral's daily energy requirements, the implications of altered metabolic processes from exposure to thermal stress found in this study on coral-Symbiodinium associations are unknown and should be considered when assessing the stability of the symbiotic relationship under future climate conditions.

  13. Genome-Wide Analysis in Brazilians Reveals Highly Differentiated Native American Genome Regions.

    PubMed

    Mychaleckyj, Josyf C; Havt, Alexandre; Nayak, Uma; Pinkerton, Relana; Farber, Emily; Concannon, Patrick; Lima, Aldo A; Guerrant, Richard L

    2017-03-01

    Despite its population, geographic size, and emerging economic importance, disproportionately little genome-scale research exists into genetic factors that predispose Brazilians to disease, or the population genetics of risk. After identification of suitable proxy populations and careful analysis of tri-continental admixture in 1,538 North-Eastern Brazilians to estimate individual ancestry and ancestral allele frequencies, we computed 400,000 genome-wide locus-specific branch length (LSBL) Fst statistics of Brazilian Amerindian ancestry compared to European and African; and a similar set of differentiation statistics for their Amerindian component compared with the closest Asian 1000 Genomes population (surprisingly, Bengalis in Bangladesh). After ranking SNPs by these statistics, we identified the top 10 highly differentiated SNPs in five genome regions in the LSBL tests of Brazilian Amerindian ancestry compared to European and African; and the top 10 SNPs in eight regions comparing their Amerindian component to the closest Asian 1000 Genomes population. We found SNPs within or proximal to the genes CIITA (rs6498115), SMC6 (rs1834619), and KLHL29 (rs2288697) were most differentiated in the Amerindian-specific branch, while SNPs in the genes ADAMTS9 (rs7631391), DOCK2 (rs77594147), SLC28A1 (rs28649017), ARHGAP5 (rs7151991), and CIITA (rs45601437) were most highly differentiated in the Asian comparison. These genes are known to influence immune function, metabolic and anthropometry traits, and embryonic development. These analyses have identified candidate genes for selection within Amerindian ancestry, and by comparison of the two analyses, those for which the differentiation may have arisen during the migration from Asia to the Americas.

  14. Transcriptomic Analysis of Thermally Stressed Symbiodinium Reveals Differential Expression of Stress and Metabolism Genes

    PubMed Central

    Gierz, Sarah L.; Forêt, Sylvain; Leggat, William

    2017-01-01

    Endosymbioses between dinoflagellate algae (Symbiodinium sp.) and scleractinian coral species form the foundation of coral reef ecosystems. The coral symbiosis is highly susceptible to elevated temperatures, resulting in coral bleaching, where the algal symbiont is released from host cells. This experiment aimed to determine the transcriptional changes in cultured Symbiodinium, to better understand the response of cellular mechanisms under future temperature conditions. Cultures were exposed to elevated temperatures (average 31°C) or control conditions (24.5°C) for a period of 28 days. Whole transcriptome sequencing of Symbiodinium cells on days 4, 19, and 28 were used to identify differentially expressed genes under thermal stress. A large number of genes representing 37.01% of the transcriptome (∼23,654 unique genes, FDR < 0.05) with differential expression were detected at no less than one of the time points. Consistent with previous studies of Symbiodinium gene expression, fold changes across the transcriptome were low, with 92.49% differentially expressed genes at ≤2-fold change. The transcriptional response included differential expression of genes encoding stress response components such as the antioxidant network and molecular chaperones, cellular components such as core photosynthesis machinery, integral light-harvesting protein complexes and enzymes such as fatty acid desaturases. Differential expression of genes encoding glyoxylate cycle enzymes were also found, representing the first report of this in Symbiodinium. As photosynthate transfer from Symbiodinium to coral hosts provides up to 90% of a coral’s daily energy requirements, the implications of altered metabolic processes from exposure to thermal stress found in this study on coral-Symbiodinium associations are unknown and should be considered when assessing the stability of the symbiotic relationship under future climate conditions. PMID:28293249

  15. Differential gene expression and alternative splicing between diploid and tetraploid watermelon.

    PubMed

    Saminathan, Thangasamy; Nimmakayala, Padma; Manohar, Sumanth; Malkaram, Sridhar; Almeida, Aldo; Cantrell, Robert; Tomason, Yan; Abburi, Lavanya; Rahman, Mohammad A; Vajja, Venkata G; Khachane, Amit; Kumar, Brajendra; Rajasimha, Harsha K; Levi, Amnon; Wehner, Todd; Reddy, Umesh K

    2015-03-01

    The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits.

  16. Effects of the neurotoxic thionophosphate pesticide chlorpyrifos on differentiating alternative models.

    PubMed

    Amaroli, Andrea; Aluigi, Maria Grazia; Falugi, Carla; Chessa, Maria Giovanna

    2013-02-01

    Studies by researchers worldwide have revealed that, even in industrialised nations, people, infants and the aged in particular, are even more exposed to neurotoxic drugs as a consequence of the increased quantity of pesticide residues in food. This phenomenon, as underlined by The Worldwatch Institute (2006), is linked to the exponential increase in the use of these toxic compounds over the last 40 years, up from 0.49 kg per hectare in 1961 to 2 kg in 2004, with the result that these substances are found in the daily diet. Many studies have demonstrated how the assumption of pesticides in the neonatal period and early infancy can alter the development and function of the nervous, immune, endocrine and reproductive apparatuses. Moreover, the unequivocal relationship between brain tumours, infant leukemia and pesticides are well recognised. On the basis of the above information, the effects of the neurotoxic thionophosphate pesticide chlorpyrifos (CPF) have been tested, considering biomarkers of toxicity and toxicity endpoint, on the biological models Dictyostelium discoideum, Paracentrotus lividus, and NTera2 Cells, as they are compatible with the 3Rs strategy (Reduction, Replacement, and Refinement in animal experiments). Our results have revealed that developing organisms are particularly sensitive to the toxic effects of CPF.

  17. Differential transcriptional regulation by alternatively designed mechanisms: A mathematical modeling approach.

    PubMed

    Yildirim, Necmettin; Aktas, Mehmet Emin; Ozcan, Seyma Nur; Akbas, Esra; Ay, Ahmet

    2016-08-02

    Cells maintain cellular homeostasis employing different regulatory mechanisms to respond external stimuli. We study two groups of signal-dependent transcriptional regulatory mechanisms. In the first group, we assume that repressor and activator proteins compete for binding to the same regulatory site on DNA (competitive mechanisms). In the second group, they can bind to different regulatory regions in a noncompetitive fashion (noncompetitive mechanisms). For both competitive and noncompetitive mechanisms, we studied the gene expression dynamics by increasing the repressor or decreasing the activator abundance (inhibition mechanisms), or by decreasing the repressor or increasing the activator abundance (activation mechanisms). We employed delay differential equation models. Our simulation results show that the competitive and noncompetitive inhibition mechanisms exhibit comparable repression effectiveness. However, response time is fastest in the noncompetitive inhibition mechanism due to increased repressor abundance, and slowest in the competitive inhibition mechanism by increased repressor level. The competitive and noncompetitive inhibition mechanisms through decreased activator abundance show comparable and moderate response times, while the competitive and noncompetitive activation mechanisms by increased activator protein level display more effective and faster response. Our study exemplifies the importance of mathematical modeling and computer simulation in the analysis of gene expression dynamics.

  18. Differential isoform expression and protein localization from alternatively spliced Apetala2 in peanut under drought stress.

    PubMed

    Park, So-Yon; Grabau, Elizabeth

    2016-11-01

    APETALA2 (AP2) belongs to the AP2/Ethylene Responsive Factor (ERF) family and regulates expression levels of downstream stress responsive genes as a transcription factor. In this study, we cloned six different isoforms of AhAP2 from peanut (Arachis hypogaea). Four isoforms (AhAP2.1, AhAP2.2, AhAP2.3 and AhAP2.4) had both AP2/ERF DNA binding domains and ERF-associated amphiphilic repression (EAR) motifs. Two isoforms (AhAP2.5 and AhAP2.6) only had an EAR suppressor domain. After agroinfiltration, AhAP2.1, AhAP2.3, and AhAP2.4 fused to yellow fluorescent protein (YFP) showed localization to the nucleolus, which is the site of transcription and ribosome biogenesis. AhAP2.2-YFP showed a dispersed signal in the nucleus. AhAP2.5 and AhAP2.6 fused to YFP localized to both the nucleus and cytoplasm. In addition, increased levels of AhAP2.1 and AhAP2.2 transcripts were observed in drought-treated peanut leaves, suggesting differential transcriptional regulation under drought stress conditions.

  19. An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians

    PubMed Central

    Forsthoefel, David J.; James, Noelle P.; Escobar, David J.; Stary, Joel M.; Vieira, Ana P.; Waters, Forrest A.; Newmark, Phillip A.

    2012-01-01

    SUMMARY Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of post-mitotic tissues. Understanding how these processes are orchestrated requires characterizing cell type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis, and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling post-embryonic organogenesis. PMID:23079596

  20. High-Throughput Proteomics Reveals the Unicellular Roots of Animal Phosphosignaling and Cell Differentiation.

    PubMed

    Sebé-Pedrós, Arnau; Peña, Marcia Ivonne; Capella-Gutiérrez, Salvador; Antó, Meritxell; Gabaldón, Toni; Ruiz-Trillo, Iñaki; Sabidó, Eduard

    2016-10-24

    Cell-specific regulation of protein levels and activity is essential for the distribution of functions among multiple cell types in animals. The finding that many genes involved in these regulatory processes have a premetazoan origin raises the intriguing possibility that the mechanisms required for spatially regulated cell differentiation evolved prior to the appearance of animals. Here, we use high-throughput proteomics in Capsaspora owczarzaki, a close unicellular relative of animals, to characterize the dynamic proteome and phosphoproteome profiles of three temporally distinct cell types in this premetazoan species. We show that life-cycle transitions are linked to extensive proteome and phosphoproteome remodeling and that they affect key genes involved in animal multicellularity, such as transcription factors and tyrosine kinases. The observation of shared features between Capsaspora and metazoans indicates that elaborate and conserved phosphosignaling and proteome regulation supported temporal cell-type differentiation in the unicellular ancestor of animals.

  1. An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians.

    PubMed

    Forsthoefel, David J; James, Noëlle P; Escobar, David J; Stary, Joel M; Vieira, Ana P; Waters, Forrest A; Newmark, Phillip A

    2012-10-16

    Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of postmitotic tissues. Understanding how these processes are orchestrated requires characterizing cell-type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling postembryonic organogenesis.

  2. Evaluating a humane alternative to the bark collar: Automated differential reinforcement of not barking in a home-alone setting.

    PubMed

    Protopopova, Alexandra; Kisten, Dmitri; Wynne, Clive

    2016-12-01

    The aim of this study was to develop a humane alternative to the traditional remote devices that deliver punishers contingent on home-alone dog barking. Specifically, we evaluated the use of remote delivery of food contingent on intervals of not barking during the pet owner's absence. In Experiment 1, 5 dogs with a history of home-alone nuisance barking were recruited. Using an ABAB reversal design, we demonstrated that contingent remote delivery of food decreased home-alone barking for 3 of the dogs. In Experiment 2, we demonstrated that it is possible to thin the differential-reinforcement-of-other-behavior (DRO) schedule gradually, resulting in a potentially more acceptable treatment. Our results benefit the dog training community by providing a humane tool to combat nuisance barking.

  3. Genetic diversity and differentiation in Dalbergia sissoo (Fabaceae) as revealed by RAPD.

    PubMed

    Wang, B-Y; Shi, L; Ruan, Z-Y; Deng, J

    2011-01-01

    Dalbergia sissoo, a wind-dispersed tropical tree, is one of the most preferred timber tree species of South Asia. Genetic diversity and differentiation among natural populations of D. sissoo were examined for the first time. We found a relatively high level of genetic diversity in D. sissoo, both at the species level (percentage of polymorphic bands = 89.11%; H = 0.2730; I = 0.4180) and the population level (percentage of polymorphic bands = 68.7%; H = 0.239; I = 0.358), along with a relatively low degree of differentiation among populations (GST = 0.1311; AMOVA = 14.69%). Strong gene flow among populations was estimated, N(m) = 3.3125. The Mantel test suggested that genetic distances between populations were weakly correlated with geographic distances (R = 0.3702, P = 0.1236). The high level of genetic diversity, low degree of differentiation, strong gene flow, and weak correlation between genetic and geographic distances can be explained by its biological character and wide-spread planting. This information will be useful for the introduction, conservation and further studies of D. sissoo and related species.

  4. Genetic diversity and differentiation in Camellia reticulata (Theaceae) polyploid complex revealed by ISSR and ploidy.

    PubMed

    Wang, B-Y; Ruan, Z-Y

    2012-03-06

    Camellia reticulata is a well-known ornamental and oil plant that is endemic to southwest China. This species shows three cell ploidies, i.e., diploidy, tetraploidy and hexaploidy. We made the first investigation of genetic diversity and differentiation of natural populations of C. reticulata, and 114 individuals from 6 populations were sampled. Cytogeography results showed that ploidy is invariable within populations and evenly distributed. A relatively high level of genetic diversity was found in C. reticulata, both at the species level (PPB = 88.89%; H = 0.2809; I = 0.4278) and at the population level (mean PPB = 42.13%; mean H = 0.14; mean I = 0.21). We found a relatively low degree of differentiation among ploidies (G(ST) = 0.2384; AMOVA = 10.26%) and a relatively high degree of differentiation among populations (G(CS) = 0.3807; AMOVA = 48.75%). The high genetic diversity can be explained by its biological character, wide distribution and ploidies, and the special genetic structure can be ascribed to polyploid origin from hybridization with different Camellia spp. This information will be useful for the introduction, conservation and further studies of C. reticulata and related species.

  5. Differential proteomics reveals novel insights into Nosema-honey bee interactions.

    PubMed

    Kurze, Christoph; Dosselli, Ryan; Grassl, Julia; Le Conte, Yves; Kryger, Per; Baer, Boris; Moritz, Robin F A

    2016-12-01

    Host manipulation is a common strategy by parasites to reduce host defense responses, enhance development, host exploitation, reproduction and, ultimately, transmission success. As these parasitic modifications can reduce host fitness, increased selection pressure may result in reciprocal adaptations of the host. Whereas the majority of studies on host manipulation have explored resistance against parasites (i.e. ability to prevent or limit an infection), data describing tolerance mechanisms (i.e. ability to limit harm of an infection) are scarce. By comparing differential protein abundance, we provide evidence of host-parasite interactions in the midgut proteomes of N. ceranae-infected and uninfected honey bees from both Nosema-tolerant and Nosema-sensitive lineages. We identified 16 proteins out of 661 protein spots that were differentially abundant between experimental groups. In general, infections of Nosema resulted in an up-regulation of the bee's energy metabolism. Additionally, we identified 8 proteins that were differentially abundant between tolerant and sensitive honey bees regardless of the Nosema infection. Those proteins were linked to metabolism, response to oxidative stress and apoptosis. In addition to bee proteins, we also identified 3 Nosema ceranae proteins. Interestingly, abundance of two of these Nosema proteins were significantly higher in infected Nosema-sensitive honeybees relative to the infected Nosema-tolerant lineage. This may provide a novel candidate for studying the molecular interplay between N. ceranae and its honey bee host in more detail.

  6. NFAT2 Isoforms Differentially Regulate Gene Expression, Cell Death, and Transformation through Alternative N-Terminal Domains

    PubMed Central

    Lucena, Pedro I.; Faget, Douglas V.; Pachulec, Emilia; Robaina, Marcela C.; Klumb, Claudete E.

    2015-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2α and NFAT2β that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2α (CA-NFAT2α) and CA-NFAT2β distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2α strongly induces cell transformation, CA-NFAT2β leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF-α). CA-NFAT2β also increases cell death and upregulates Fas ligand (FasL) and TNF-α in CD4+ T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2β-specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2α isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions. PMID:26483414

  7. NFAT2 Isoforms Differentially Regulate Gene Expression, Cell Death, and Transformation through Alternative N-Terminal Domains.

    PubMed

    Lucena, Pedro I; Faget, Douglas V; Pachulec, Emilia; Robaina, Marcela C; Klumb, Claudete E; Robbs, Bruno K; Viola, João P B

    2016-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2α and NFAT2β that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2α (CA-NFAT2α) and CA-NFAT2β distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2α strongly induces cell transformation, CA-NFAT2β leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF-α). CA-NFAT2β also increases cell death and upregulates Fas ligand (FasL) and TNF-α in CD4(+) T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2β-specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2α isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions.

  8. Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor▿†

    PubMed Central

    Newton, Ryan J.; VandeWalle, Jessica L.; Borchardt, Mark A.; Gorelick, Marc H.; McLellan, Sandra L.

    2011-01-01

    The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the Lachnospiraceae family, which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci and the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to

  9. Alternative splicing and differential expression of two transcripts of nicotine adenine dinucleotide phosphate oxidase B gene from Zea mays.

    PubMed

    Lin, Fan; Zhang, Yun; Jiang, Ming-Yi

    2009-03-01

    With the exception of rice, little is known about the existence of respiratory burst oxidase homolog (rboh) gene in cereals. The present study reports the cloning and analysis of a novel rboh gene, termed ZmrbohB, from maize (Zea mays L.). The full-length cDNA of ZmrbohB encodes a 942 amino acid protein containing all of the respiratory burst oxidase homolog catalytically critical motifs. Alternative splicing of ZmrbohB has generated two transcript isoforms, ZmrbohB-alpha and -beta. Spliced transcript ZmrbohB-beta retains an unspliced intron 11 that carries a premature termination codon and probably leads to nonsense-mediated mRNA decay. Expression analysis showed that two splice isoforms were differentially expressed in various tissues and at different developmental stages, and the major product was ZmrbohB-alpha. The transcripts of ZmrbohB-alpha accumulated markedly when the maize seedlings were subjected to various abiotic stimuli, such as wounding, cold (4 degrees C), heat (40 degrees C), UV and salinity stress. In addition, several abiotic stimuli also affected the alternative splicing pattern of ZmrbohB except wounding. These results provide new insight into roles in the expression regulation of plant rboh genes and suggest that ZmrbohB gene may play a role in response to environmental stresses.

  10. A phased strategy to differentiate human CD14+monocytes into classically and alternatively activated macrophages and dendritic cells.

    PubMed

    Zarif, Jelani C; Hernandez, James R; Verdone, James E; Campbell, Scott P; Drake, Charles G; Pienta, Kenneth J

    2016-01-01

    There are currently several in vitro strategies to differentiate human CD14(+) monocytes isolated from peripheral blood mononuclear cells (PBMCs) into the M1 or M2 macrophage cell types. Each cell type is then verified using flow cytometric analysis of cell-surface markers. Human CD14(+) monocytes have the potential to differentiate into M1 and M2 macrophages, both of which demonstrate varying degrees of cell-surface antigen overlap. Using multiple surface markers with current macrophage polarization protocols, our data reveal several limitations of currently used methods, such as highly ambiguous cell types that possess cell-surface marker overlap and functional similarities. Utilizing interleukin-6 (IL-6) and two phases of cytokine exposure, we have developed a protocol to differentiate human monocytes into M1, M2, or dendritic cells (DCs) with greater efficiency and fidelity relative to macrophages and DCs that are produced by commonly used methods. This is achieved via alterations in cytokine composition, dosing, and incubation times, as well as improvements in verification methodology. Our method reliably reproduces human in vitro monocyte-derived DCs and macrophage models that will aid in better defining and understanding innate and adaptive immunity, as well as pathologic states.

  11. Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

    SciTech Connect

    Sukseree, Supawadee; Rossiter, Heidemarie; Mildner, Michael; Pammer, Johannes; Buchberger, Maria; Gruber, Florian; Watanapokasin, Ramida; Tschachler, Erwin; Eckhart, Leopold

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. Black-Right-Pointing-Pointer Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. Black-Right-Pointing-Pointer Autophagy was required for normal terminal differentiation of preputial gland cells. Black-Right-Pointing-Pointer Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

  12. Quantitative proteomic analysis to decipher the differential apoptotic response of bortezomib-treated APL cells before and after retinoic acid differentiation reveals involvement of protein toxicity mechanisms.

    PubMed

    Uttenweiler-Joseph, Sandrine; Bouyssié, David; Calligaris, David; Lutz, Pierre G; Monsarrat, Bernard; Burlet-Schiltz, Odile

    2013-01-01

    The ubiquitin-proteasome system allows the targeted degradation of proteins and plays a critical role in the regulation of many cellular processes. Proteasome inhibition is a recent antitumor therapeutic strategy and bortezomib was the first proteasome inhibitor approved for clinical use. In this study, we used the NB4 cell line to investigate the effects of bortezomib toward acute promyelocytic leukemia cells before and after retinoic acid-induced differentiation. We showed that apoptosis level after bortezomib treatment is higher in NB4 cells than in differentiated NB4 cells. To compare early protein variations upon bortezomib treatment in both NB4 cell populations, we performed a quantitative proteomic analysis based on iTRAQ peptide labeling followed by data analysis with in-house developed scripts. This strategy revealed the regulation of 14 proteins principally involved in protein stress response and apoptosis in NB4 cells after proteasome inhibition. Altogether, our results suggest that the differential level of apoptosis induced by bortezomib treatment in both NB4 cell populations could result from distinct protein toxicity level.

  13. Transcriptomic Analysis Reveals Mechanisms of Sterile and Fertile Flower Differentiation and Development in Viburnum macrocephalum f. keteleeri

    PubMed Central

    Lu, Zhaogeng; Xu, Jing; Li, Weixing; Zhang, Li; Cui, Jiawen; He, Qingsong; Wang, Li; Jin, Biao

    2017-01-01

    Sterile and fertile flowers are an important evolutionary developmental (evo-devo) phenotype in angiosperm flowers, playing important roles in pollinator attraction and sexual reproductive success. However, the gene regulatory mechanisms underlying fertile and sterile flower differentiation and development remain largely unknown. Viburnum macrocephalum f. keteleeri, which possesses fertile and sterile flowers in a single inflorescence, is a useful candidate species for investigating the regulatory networks in differentiation and development. We developed a de novo-assembled flower reference transcriptome. Using RNA sequencing (RNA-seq), we compared the expression patterns of fertile and sterile flowers isolated from the same inflorescence over its rapid developmental stages. The flower reference transcriptome consisted of 105,683 non-redundant transcripts, of which 5,675 transcripts showed significant differential expression between fertile and sterile flowers. Combined with morphological and cytological changes between fertile and sterile flowers, we identified expression changes of many genes potentially involved in reproductive processes, phytohormone signaling, and cell proliferation and expansion using RNA-seq and qRT-PCR. In particular, many transcription factors (TFs), including MADS-box family members and ABCDE-class genes, were identified, and expression changes in TFs involved in multiple functions were analyzed and highlighted to determine their roles in regulating fertile and sterile flower differentiation and development. Our large-scale transcriptional analysis of fertile and sterile flowers revealed the dynamics of transcriptional networks and potentially key components in regulating differentiation and development of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri. Our data provide a useful resource for Viburnum transcriptional research and offer insights into gene regulation of differentiation of diverse evo-devo processes in

  14. Transcriptomic Analysis Reveals Mechanisms of Sterile and Fertile Flower Differentiation and Development in Viburnum macrocephalum f. keteleeri.

    PubMed

    Lu, Zhaogeng; Xu, Jing; Li, Weixing; Zhang, Li; Cui, Jiawen; He, Qingsong; Wang, Li; Jin, Biao

    2017-01-01

    Sterile and fertile flowers are an important evolutionary developmental (evo-devo) phenotype in angiosperm flowers, playing important roles in pollinator attraction and sexual reproductive success. However, the gene regulatory mechanisms underlying fertile and sterile flower differentiation and development remain largely unknown. Viburnum macrocephalum f. keteleeri, which possesses fertile and sterile flowers in a single inflorescence, is a useful candidate species for investigating the regulatory networks in differentiation and development. We developed a de novo-assembled flower reference transcriptome. Using RNA sequencing (RNA-seq), we compared the expression patterns of fertile and sterile flowers isolated from the same inflorescence over its rapid developmental stages. The flower reference transcriptome consisted of 105,683 non-redundant transcripts, of which 5,675 transcripts showed significant differential expression between fertile and sterile flowers. Combined with morphological and cytological changes between fertile and sterile flowers, we identified expression changes of many genes potentially involved in reproductive processes, phytohormone signaling, and cell proliferation and expansion using RNA-seq and qRT-PCR. In particular, many transcription factors (TFs), including MADS-box family members and ABCDE-class genes, were identified, and expression changes in TFs involved in multiple functions were analyzed and highlighted to determine their roles in regulating fertile and sterile flower differentiation and development. Our large-scale transcriptional analysis of fertile and sterile flowers revealed the dynamics of transcriptional networks and potentially key components in regulating differentiation and development of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri. Our data provide a useful resource for Viburnum transcriptional research and offer insights into gene regulation of differentiation of diverse evo-devo processes in

  15. Genome-wide view of genetic diversity reveals paths of selection and cultivar differentiation in peach domestication

    PubMed Central

    Akagi, Takashi; Hanada, Toshio; Yaegaki, Hideaki; Gradziel, Thomas M.; Tao, Ryutaro

    2016-01-01

    Domestication and cultivar differentiation are requisite processes for establishing cultivated crops. These processes inherently involve substantial changes in population structure, including those from artificial selection of key genes. In this study, accessions of peach (Prunus persica) and its wild relatives were analysed genome-wide to identify changes in genetic structures and gene selections associated with their differentiation. Analysis of genome-wide informative single-nucleotide polymorphism loci revealed distinct changes in genetic structures and delineations among domesticated peach and its wild relatives and among peach landraces and modern fruit (F) and modern ornamental (O-A) cultivars. Indications of distinct changes in linkage disequilibrium extension/decay and of strong population bottlenecks or inbreeding were identified. Site frequency spectrum- and extended haplotype homozygosity-based evaluation of genome-wide genetic diversities supported selective sweeps distinguishing the domesticated peach from its wild relatives and each F/O-A cluster from the landrace clusters. The regions with strong selective sweeps harboured promising candidates for genes subjected to selection. Further sequence-based evaluation further defined the candidates and revealed their characteristics. All results suggest opportunities for identifying critical genes associated with each differentiation by analysing genome-wide genetic diversity in currently established populations. This approach obviates the special development of genetic populations, which is particularly difficult for long-lived tree crops. PMID:27085183

  16. Genome-wide view of genetic diversity reveals paths of selection and cultivar differentiation in peach domestication.

    PubMed

    Akagi, Takashi; Hanada, Toshio; Yaegaki, Hideaki; Gradziel, Thomas M; Tao, Ryutaro

    2016-06-01

    Domestication and cultivar differentiation are requisite processes for establishing cultivated crops. These processes inherently involve substantial changes in population structure, including those from artificial selection of key genes. In this study, accessions of peach (Prunus persica) and its wild relatives were analysed genome-wide to identify changes in genetic structures and gene selections associated with their differentiation. Analysis of genome-wide informative single-nucleotide polymorphism loci revealed distinct changes in genetic structures and delineations among domesticated peach and its wild relatives and among peach landraces and modern fruit (F) and modern ornamental (O-A) cultivars. Indications of distinct changes in linkage disequilibrium extension/decay and of strong population bottlenecks or inbreeding were identified. Site frequency spectrum- and extended haplotype homozygosity-based evaluation of genome-wide genetic diversities supported selective sweeps distinguishing the domesticated peach from its wild relatives and each F/O-A cluster from the landrace clusters. The regions with strong selective sweeps harboured promising candidates for genes subjected to selection. Further sequence-based evaluation further defined the candidates and revealed their characteristics. All results suggest opportunities for identifying critical genes associated with each differentiation by analysing genome-wide genetic diversity in currently established populations. This approach obviates the special development of genetic populations, which is particularly difficult for long-lived tree crops.

  17. RNA sequencing reveals small RNAs differentially expressed between incipient Japanese threespine sticklebacks

    PubMed Central

    2013-01-01

    Background Non-coding small RNAs, ranging from 20 to 30 nucleotides in length, mediate the regulation of gene expression and play important roles in many biological processes. One class of small RNAs, microRNAs (miRNAs), are highly conserved across taxa and mediate the regulation of the chromatin state and the post-transcriptional regulation of messenger RNA (mRNA). Another class of small RNAs is the Piwi-interacting RNAs, which play important roles in the silencing of transposons and other functional genes. Although the biological functions of the different small RNAs have been elucidated in several laboratory animals, little is known regarding naturally occurring variation in small RNA transcriptomes among closely related species. Results We employed next-generation sequencing technology to compare the expression profiles of brain small RNAs between sympatric species of the Japanese threespine stickleback (Gasterosteus aculeatus). We identified several small RNAs that were differentially expressed between sympatric Pacific Ocean and Japan Sea sticklebacks. Potential targets of several small RNAs were identified as repetitive sequences. Female-biased miRNA expression from the old X chromosome was also observed, and it was attributed to the degeneration of the Y chromosome. Conclusions Our results suggest that expression patterns of small RNA can differ between incipient species and may be a potential mechanism underlying differential mRNA expression and transposon activity. PMID:23547919

  18. Large Scale Immune Profiling of Infected Humans and Goats Reveals Differential Recognition of Brucella melitensis Antigens

    PubMed Central

    Liang, Li; Leng, Diana; Burk, Chad; Nakajima-Sasaki, Rie; Kayala, Matthew A.; Atluri, Vidya L.; Pablo, Jozelyn; Unal, Berkay; Ficht, Thomas A.; Gotuzzo, Eduardo; Saito, Mayuko; Morrow, W. John W.; Liang, Xiaowu; Baldi, Pierre; Gilman, Robert H.; Vinetz, Joseph M.; Tsolis, Renée M.; Felgner, Philip L.

    2010-01-01

    Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host. PMID:20454614

  19. Genetic diversity and differentiation of the Ryukyu endemic frog Babina holsti as revealed by mitochondrial DNA.

    PubMed

    Tominaga, Atsushi; Matsui, Masafumi; Nakata, Katsushi

    2014-02-01

    We surveyed the genetic diversity and genetic differentiation of an endangered frog, Babina holsti, endemic to Okinawajima and Tokashikijima Islands of the Ryukyus, to elucidate its divergence history and obtain basic data for its conservation. Genetic differentiation between the two island lineages is moderate (3.1% p-distance in the cyt b gene). This result suggests that the two island lineages have been isolated between the late Pliocene and the middle Pleistocene and have never migrated between the current northern part of Okinawajima and Tokashikijima Islands, which were once connected in the late Pleistocene glacial age. On Okinawajima Island, the southernmost sample was constituted by a unique haplotype, without considerable genetic distance from haplotypes detected from northern samples. This unique haplotype composition in the southernmost sample would have resulted from the restricted gene flow between the southernmost population and the other populations in Okinawajima Island. Furthermore, the absence of genetic diversity within the southernmost sample indicates that this population has recently experienced population size reduction, possibly by predation pressure from an introduced mongoose, which is more abundant in the southern part than in the northern part of the island. Lower genetic diversity in the Tokashikijima sample implies a small effective population size for mitochondrial DNA (mtDNA) in B. holsti on the island. Immediate conservation measures should be taken for the populations from the southernmost range in Okinawajima and Tokashikijima.

  20. Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies.

    PubMed

    Sakowski, Stacey A; Geddes, Timothy J; Thomas, David M; Levi, Edi; Hatfield, James S; Kuhn, Donald M

    2006-04-26

    Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.

  1. Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation

    PubMed Central

    Carthy, Jon M.; Stöter, Martin; Bellomo, Claudia; Vanlandewijck, Michael; Heldin, Angelos; Morén, Anita; Kardassis, Dimitris; Gahman, Timothy C.; Shiau, Andrew K.; Bickle, Marc; Zerial, Marino; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition. In addition to TGF-β receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-β-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-β-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer. PMID:27430378

  2. Pooled ecotype sequencing reveals candidate genetic mechanisms for adaptive differentiation and reproductive isolation.

    PubMed

    Gould, Billie A; Chen, Yani; Lowry, David B

    2017-01-01

    The early stages of speciation are often characterized by the formation of partially reproductively isolated ecotypes, which evolve as a by-product of divergent selective forces that are endemic to different habitats. Identifying the genomic regions, genes and ultimately functional polymorphisms that are involved in the processes of ecotype formation is inherently challenging, as there are likely to be many different loci involved in the process. To localize candidate regions of the genome contributing to ecotype formation, we conducted whole-genome pooled sequencing (pool-seq) with 47 coastal perennial and 50 inland annual populations of the yellow monkeyflower, Mimulus guttatus. Coastal perennial and inland annual ecotypes of M. guttatus have previously been shown to be ecologically reproductively isolated and highly locally adapted to their respective habitats. Our pool-seq results found allelic differentiation between the ecotypes for two chromosomal inversions, suggesting that frequencies of inversion heterokaryotypes are strongly differentiated between the ecotypes. Further, there were elevated levels of nonsynonymous change across chromosomal inversions. Across the genome, we identified multiple strong candidate genes potentially driving the morphological, life history and salt tolerance differences between the ecotypes. Several candidate genes coincide with previously identified quantitative trait locus regions and also show a signature of recent natural selection. Overall, the results of our study add to growing support for a major role of chromosomal inversions in adaptation and speciation and provide new insights into the genetic mechanisms underlying classic plant ecotype adaptations to wet and dry habitats.

  3. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation

    PubMed Central

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J.; Zhang, Jianyi; Ge, Ying

    2015-01-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function. PMID:26033914

  4. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

    PubMed

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J; Zhang, Jianyi; Ge, Ying

    2015-08-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.

  5. Large scale immune profiling of infected humans and goats reveals differential recognition of Brucella melitensis antigens.

    PubMed

    Liang, Li; Leng, Diana; Burk, Chad; Nakajima-Sasaki, Rie; Kayala, Matthew A; Atluri, Vidya L; Pablo, Jozelyn; Unal, Berkay; Ficht, Thomas A; Gotuzzo, Eduardo; Saito, Mayuko; Morrow, W John W; Liang, Xiaowu; Baldi, Pierre; Gilman, Robert H; Vinetz, Joseph M; Tsolis, Renée M; Felgner, Philip L

    2010-05-04

    Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

  6. Differential proteins of the optic ganglion in octopus vulgaris under methanol stress revealed using proteomics.

    PubMed

    Huang, Lin; Huang, Qing-Yu; Chen, Hai-Bin; Huang, Fu-Sheng; Huang, He-Qing

    2011-10-01

    An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.

  7. Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes

    SciTech Connect

    Hawley, Alyse K.; Brewer, Heather M.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Hallam, Steven J.

    2014-08-05

    Oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified marine waters. Currently OMZs are expanding due to global climate change. This expansion alters marine ecosystem function and the productivity of fisheries due to habitat compression and changes in biogeochemical cycling leading to fixed nitrogen loss and greenhouse gas production. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally anoxic fjord, Saanich Inlet to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification and inorganic carbon fixation predominantly co-varied with abundance and distribution patterns of Thaumarchaeota, Nitrospira, Planctomycetes and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Within these groups, pathways mediating inorganic carbon fixation and nitrogen and sulfur transformations were differentially expressed across the redoxcline. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters and denitrification, sulfur-oxidation and inorganic carbon fixation pathways affiliated with SUP05 dominated suboxic and anoxic waters. Nitrite-oxidation and anammox pathways affiliated with Nitrospina and Planctomycetes respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The differential expression of these pathways under changing water column redox conditions has quantitative implications for coupled biogeochemical cycling linking different modes of inorganic carbon fixation with distributed nitrogen and sulfur-based energy metabolism extensible to coastal and open ocean OMZs.

  8. Comparative metagenomics reveals microbial community differentiation in a biological heap leaching system.

    PubMed

    Hu, Qi; Guo, Xue; Liang, Yili; Hao, Xiaodong; Ma, Liyuan; Yin, Huaqun; Liu, Xueduan

    2015-01-01

    The microbial community in a biological heap leaching (BHL) system is crucial for the decomposition of ores. However, the microbial community structure and functional differentiation in different parts of a biological heap leaching system are still unknown. In this study, metagenomic sequencing was used to fully illuminate the microbial community differentiation in the pregnant leach solution (PLS) and leaching heap (LH) of a BHL system. Long-read sequences (1.3 million) were obtained for the two samples, and the MG_RAST server was used to perform further analysis. The taxa analysis results indicated that the dominant genera of PLS is autotrophic bacterium Acidithiobacillus, but heterotrophic bacterium Acidiphilium is predominant in LH. Furthermore, functional annotation and hierarchical comparison with different reference samples showed that the abundant presence of genes was involved in transposition, DNA repair and heavy metal transport. The sequences related to transposase, which is important for the survival of the organism in the hostile environment, were both mainly classified into Acidiphilium for PLS and LH. These results indicated that not only autotrophic bacteria such as Acidithiobacillus, but also heterotrophic bacteria such as Acidiphilium, were essential participants in the bioleaching process. This new meta-view research will further facilitate the effective application of bioleaching.

  9. Transcriptome analysis of differentiating trypanosomes reveals the existence of multiple post-transcriptional regulons

    PubMed Central

    Queiroz, Rafael; Benz, Corinna; Fellenberg, Kurt; Hoheisel, Jörg D; Clayton, Christine

    2009-01-01

    Background Trypanosome gene expression is regulated almost exclusively at the post-transcriptional level, with mRNA degradation playing a decisive role. When trypanosomes are transferred from the blood of a mammal to the midgut of a Tsetse fly, they transform to procyclic forms: gene expression is reprogrammed, changing the cell surface and switching the mode of energy metabolism. Within the blood, trypanosomes can pre-adapt for Tsetse transmission, becoming growth-arrested stumpy forms. We describe here the transitions in gene expression that occur during differentiation of in-vitro cultured bloodstream forms to procyclic forms. Results Some mRNAs showed changes within 30 min of cis-aconitate addition, whereas others responded 12-24 hours later. For the first 12 h after addition of cis-aconitate, cells accumulated at the G1 phase of the cell cycle, and showed decreases in mRNAs required for proliferation, mimicking the changes seen in stumpy forms: many mRNAs needed for ribosomal and flagellar biogenesis showed striking co-regulation. Other mRNAs encoding components of signal transduction pathways and potential regulators were specifically induced only during differentiation. Messenger RNAs encoding proteins required for individual metabolic pathways were often co-regulated. Conclusion Trypanosome genes form post-transcriptional regulons in which mRNAs with functions in particular pathways, or encoding components of protein complexes, show almost identical patterns of regulation. PMID:19857263

  10. Down Syndrome Developmental Brain Transcriptome Reveals Defective Oligodendrocyte Differentiation and Myelination.

    PubMed

    Olmos-Serrano, Jose Luis; Kang, Hyo Jung; Tyler, William A; Silbereis, John C; Cheng, Feng; Zhu, Ying; Pletikos, Mihovil; Jankovic-Rapan, Lucija; Cramer, Nathan P; Galdzicki, Zygmunt; Goodliffe, Joseph; Peters, Alan; Sethares, Claire; Delalle, Ivana; Golden, Jeffrey A; Haydar, Tarik F; Sestan, Nenad

    2016-03-16

    Trisomy 21, or Down syndrome (DS), is the most common genetic cause of developmental delay and intellectual disability. To gain insight into the underlying molecular and cellular pathogenesis, we conducted a multi-region transcriptome analysis of DS and euploid control brains spanning from mid-fetal development to adulthood. We found genome-wide alterations in the expression of a large number of genes, many of which exhibited temporal and spatial specificity and were associated with distinct biological processes. In particular, we uncovered co-dysregulation of genes associated with oligodendrocyte differentiation and myelination that were validated via cross-species comparison to Ts65Dn trisomy mice. Furthermore, we show that hypomyelination present in Ts65Dn mice is in part due to cell-autonomous effects of trisomy on oligodendrocyte differentiation and results in slower neocortical action potential transmission. Together, these results identify defects in white matter development and function in DS, and they provide a transcriptional framework for further investigating DS neuropathogenesis.

  11. Proteomic analysis of Clostridium thermocellum ATCC 27405 reveals the upregulation of an alternative transhydrogenase-malate pathway and nitrogen assimilation in cells grown on cellulose.

    PubMed

    Burton, Euan; Martin, Vincent J J

    2012-12-01

    Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the ability to directly convert cellulosic biomass into useful products such as ethanol and hydrogen. In this study, a quantitative comparative proteomic analysis of the organism was performed to identify proteins and biochemical pathways that are differentially utilized by the organism after growth on cellobiose or cellulose. The cytoplasmic and membrane proteomes of C. thermocellum grown on cellulose or cellobiose were quantitatively compared using a metabolic (15)N isotope labelling method in conjunction with nanoLC-ESI-MS/MS (liquid chromatography - electrospray ionization - tandem mass spectrometry). In total, 1255 proteins were identified in the study, and 129 of those were able to have their relative abundance per cell compared in at least one cellular compartment in response to the substrate provided. This study reveals that cells grown on cellulose increase their abundance of phosphoenolpyruvate carboxykinase while decreasing the abundance of pyruvate dikinase and oxaloacetate decarboxylase, suggesting that the organism diverts carbon flow into a transhydrogenase-malate pathway that can increase the production of the biosynthetic intermediates NADPH and GTP. Glutamate dehydrogenase was also found to have increased abundance in cellulose-grown cells, suggesting that the assimilation of ammonia is upregulated in cells grown on the cellulosic substrates. The results illustrate a mechanism by which C. thermocellum can divert carbon into alternative pathways for the purpose of producing biosynthetic intermediates necessary to respond to growth on cellulose, including transhydrogenation of NADH to NADPH and increased nitrogen assimilation.

  12. Early transcriptional and epigenetic regulation of CD8(+) T cell differentiation revealed by single-cell RNA sequencing.

    PubMed

    Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E; He, Zhaoren; Aigner, Stefan; Metz, Patrick J; Yu, Bingfei; Wehrens, Ellen J; Lopez, Justine; Kim, Stephanie H; Zuniga, Elina I; Goldrath, Ananda W; Chang, John T; Yeo, Gene W

    2017-04-01

    During microbial infection, responding CD8(+) T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA-sequencing approach and analyzed individual CD8(+) T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants that controlled the fate specification of CD8(+) T lymphocytes. Our findings suggest a model for the differentiation of terminal effector cells initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, which highlights the power and necessity of single-cell approaches.

  13. RAPID-COMMUNICATION Genetic diversity and differentiation in natural populations of Arapaima gigas from lower Amazon revealed by microsatellites.

    PubMed

    Fazzi-Gomes, P F; Melo, N; Palheta, G; Guerreiro, S; Amador, M; Ribeiro-Dos-Santos, A K; Santos, S; Hamoy, I

    2017-02-08

    Genetic variability is one of the important criteria for species conservation decisions. This study aimed to analyze the genetic diversity and the population differentiation of two natural populations of Arapaima gigas, a species with a long history of being commercially exploited. We collected 87 samples of A. gigas from Grande Curuai Lake and Paru Lake, located in the Lower Amazon region of Amazônia, Brazil, and genotyped these samples using a multiplex panel of microsatellite markers. Our results showed that the populations of A. gigas analyzed had high levels of genetic variability, which were similar to those described in previous studies. These two populations had a significant population differentiation supported by the estimates of FST and RST (0.06), by Bayesian analysis (K = 2), and by population assignment tests, which revealed a moderate genetic distance.

  14. Differentiation processes in FeO-rich asteroids revealed by the achondrite Lewis Cliff 88763

    NASA Astrophysics Data System (ADS)

    Day, James M. D.; Corder, Christopher A.; Rumble, Douglas; Assayag, Nelly; Cartigny, Pierre; Taylor, Lawrence A.

    2015-10-01

    Olivine-dominated (70-80 modal %) achondrite meteorite Lewis Cliff (LEW) 88763 originated from metamorphism and limited partial melting of a FeO-rich parent body. The meteorite experienced some alteration on Earth, evident from subchondritic Re/Os, and redistribution of rhenium within the sample. LEW 88763 is texturally similar to winonaites, has a Δ17O value of -1.19 ± 0.10‰, and low bulk-rock Mg/(Mg+Fe) (0.39), similar to the FeO-rich cumulate achondrite Northwest Africa (NWA) 6693. The similar bulk-rock major-, minor-, and trace-element abundances of LEW 88763, relative to some carbonaceous chondrites, including ratios of Pd/Os, Pt/Os, Ir/Os, and 187Os/188Os (0.1262), implies a FeO- and volatile-rich precursor composition. Lack of fractionation of the rare earth elements, but a factor of approximately two lower highly siderophile element abundances in LEW 88763, compared with chondrites, implies limited loss of Fe-Ni-S melts during metamorphism and anatexis. These results support the generation of high Fe/Mg, sulfide, and/or metal-rich partial melts from FeO-rich parent bodies during partial melting. In detail, however, LEW 88763 cannot be a parent composition to any other meteorite sample, due to highly limited silicate melt loss (0 to <<5%). As such, LEW 88763 represents the least-modified FeO-rich achondrite source composition recognized to date and is distinct from all other meteorites. LEW 88763 should be reclassified as an anomalous achondrite that experienced limited Fe,Ni-FeS melt loss. Lewis Cliff 88763, combined with a growing collection of FeO-rich meteorites, such as brachinites, brachinite-like achondrites, the Graves Nunataks (GRA) 06128/9 meteorites, NWA 6693, and Tafassasset, has important implications for understanding the initiation of planetary differentiation. Specifically, regardless of precursor compositions, partial melting and differentiation processes appear to be similar on asteroidal bodies spanning a range of initial oxidation

  15. Comparative glandular trichome transcriptome based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species.

    PubMed

    Akhtar, Md Qussen; Qamar, Nida; Yadav, Pallavi; Kulkarni, Pallavi; Kumar, Ajay; Shasany, Ajit Kumar

    2017-02-11

    The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome was carried out. In addition to the MVA and MEP pathway genes, about 210 and 196 different terpene synthases (TPS) transcripts were identified from annotation in M. arvensis and M. × piperita, respectively, and correlated to several monoterpenes present in the essential oil. Six isoforms of (-)-menthol dehydrogenases (MD), the last enzyme of the menthol biosynthetic pathway, were identified, cloned and characterized from the transcriptome data (3 from each species). Varied expression levels and differential enzyme kinetics of these isoforms indicated the nature and composition of the product, as these isoforms generate both (-)-menthol and (+)-neomenthol from (-)-menthone and converts (-)-menthol to (-)-menthone in the reverse reaction, and hence together determine the quantity of (-)-menthol in the essential oil in these two species. Several genes for high value minor monoterpenes could also be identified from the transcriptome data. Abbreviations - AACT, acetyl-CoA C-acetyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; DGE, digital gene expression; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; FPPS, farnesyl pyrophosphate synthase; GC, gas chromatography; GPPS, geranyl pyrophosphate synthase; GT, glandular trichome; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; HMGS, hydroxymethylglutaryl-CoA synthase; IDI, isopentenyl-diphosphate delta-isomerase; IPD, isopiperitenol dehydrogenase; IPI

  16. Alternatively spliced transcripts of the thymus-specific protease PRSS16 are differentially expressed in human thymus.

    PubMed

    Luther, C; Wienhold, W; Oehlmann, R; Heinemann, M K; Melms, A; Tolosa, E

    2005-02-01

    The putative serine protease PRSS16 is abundantly expressed in the thymic cortex and the gene is encoded within the HLA I complex. Although its function is not yet defined, the very restricted expression points to a role in T-cell development in the thymus. In this study, we show that the PRSS16 mRNA is alternatively spliced to generate at least five transcripts. Apart from the full-length sequence, we found two other isoforms with all putative active site residues of the serine protease, suggesting that those variants may also be functional. Semi-quantitative analysis of the splice variants in different tissue samples revealed a strong correlation between the specific formation of alternatively spliced PRSS16 transcripts and the age and thymus pathology status of the donor. Newborn thymi express mostly the PRSS16-4 and -5 isoforms and lack the PRSS16-1 transcript, which appears around 2 years of age and stays until adulthood. Incidentally, thymi from myasthenia gravis (MG) patients with thymoma showed a marked decrease in the expression of the full-length PRSS16-1 and increased expression of the smaller isoforms. The data suggest a potential role of the PRSS16 isoforms in the postnatal morphogenesis of the thymus and in the thymus pathology related to MG.

  17. Mice lacking WRB reveal differential biogenesis requirements of tail-anchored proteins in vivo

    PubMed Central

    Rivera-Monroy, Jhon; Musiol, Lena; Unthan-Fechner, Kirsten; Farkas, Ákos; Clancy, Anne; Coy-Vergara, Javier; Weill, Uri; Gockel, Sarah; Lin, Shuh-Yow; Corey, David P.; Kohl, Tobias; Ströbel, Philipp; Schuldiner, Maya; Schwappach, Blanche; Vilardi, Fabio

    2016-01-01

    Tail-anchored (TA) proteins are post-translationally inserted into membranes. The TRC40 pathway targets TA proteins to the endoplasmic reticulum via a receptor comprised of WRB and CAML. TRC40 pathway clients have been identified using in vitro assays, however, the relevance of the TRC40 pathway in vivo remains unknown. We followed the fate of TA proteins in two tissue-specific WRB knockout mouse models and found that their dependence on the TRC40 pathway in vitro did not predict their reaction to receptor depletion in vivo. The SNARE syntaxin 5 (Stx5) was extremely sensitive to disruption of the TRC40 pathway. Screening yeast TA proteins with mammalian homologues, we show that the particular sensitivity of Stx5 is conserved, possibly due to aggregation propensity of its cytoplasmic domain. We establish that Stx5 is an autophagy target that is inefficiently membrane-targeted by alternative pathways. Our results highlight an intimate relationship between the TRC40 pathway and cellular proteostasis. PMID:28000760

  18. Meta-Analytic Connectivity Modeling Reveals Differential Functional Connectivity of the Medial and Lateral Orbitofrontal Cortex

    PubMed Central

    Zald, David H.; McHugo, Maureen; Ray, Kimberly L.; Glahn, David C.; Eickhoff, Simon B.; Laird, Angela R.

    2014-01-01

    The orbitofrontal cortex (OFC) is implicated in a broad range of behaviors and neuropsychiatric disorders. Anatomical tracing studies in nonhuman primates reveal differences in connectivity across subregions of the OFC, but data on the connectivity of the human OFC remain limited. We applied meta-analytic connectivity modeling in order to examine which brain regions are most frequently coactivated with the medial and lateral portions of the OFC in published functional neuroimaging studies. The analysis revealed a clear divergence in the pattern of connectivity for the medial OFC (mOFC) and lateral OFC (lOFC) regions. The lOFC showed coactivations with a network of prefrontal regions and areas involved in cognitive functions including language and memory. In contrast, the mOFC showed connectivity with default mode, autonomic, and limbic regions. Convergent patterns of coactivations were observed in the amygdala, hippocampus, striatum, and thalamus. A small number of regions showed connectivity specific to the anterior or posterior sectors of the OFC. Task domains involving memory, semantic processing, face processing, and reward were additionally analyzed in order to identify the different patterns of OFC functional connectivity associated with specific cognitive and affective processes. These data provide a framework for understanding the human OFC's position within widespread functional networks. PMID:23042731

  19. Comparative Circadian Metabolomics Reveal Differential Effects of Nutritional Challenge in the Serum and Liver*

    PubMed Central

    Abbondante, Serena; Eckel-Mahan, Kristin L.; Ceglia, Nicholas J.; Baldi, Pierre; Sassone-Corsi, Paolo

    2016-01-01

    Diagnosis and therapeutic interventions in pathological conditions rely upon clinical monitoring of key metabolites in the serum. Recent studies show that a wide range of metabolic pathways are controlled by circadian rhythms whose oscillation is affected by nutritional challenges, underscoring the importance of assessing a temporal window for clinical testing and thereby questioning the accuracy of the reading of critical pathological markers in circulation. We have been interested in studying the communication between peripheral tissues under metabolic homeostasis perturbation. Here we present a comparative circadian metabolomic analysis on serum and liver in mice under high fat diet. Our data reveal that the nutritional challenge induces a loss of serum metabolite rhythmicity compared with liver, indicating a circadian misalignment between the tissues analyzed. Importantly, our results show that the levels of serum metabolites do not reflect the circadian liver metabolic signature or the effect of nutritional challenge. This notion reveals the possibility that misleading reads of metabolites in circulation may result in misdiagnosis and improper treatments. Our findings also demonstrate a tissue-specific and time-dependent disruption of metabolic homeostasis in response to altered nutrition. PMID:26644470

  20. Cerebral hemovelocity reveals differential resource allocation strategies for extraverts and introverts during vigilance.

    PubMed

    Shaw, Tyler H; Nguyen, Cynthia; Satterfield, Kelly; Ramirez, Raul; McKnight, Patrick E

    2016-02-01

    Extraversion--one of the Big 5 personality factors--correlates negatively with vigilance, but most studies focus on performance outcomes and not the performance process. Previous research has shown that transcranial Doppler sonography (TCD), which measures cerebral blood flow velocity (CBFV), can be used to examine resource allocation strategies during vigilance performance. Hence, this study was designed to assess the attentional resource allocation strategies of introverts and extraverts using the CBFV measure. Twelve extroverts and 13 introverts monitored a 60-min vigilance task for a critical signal--the absence of a line on a five-circle array. The results revealed an overall performance decrement that was not modulated by extraversion. We observed an interaction between extraversion and time; CBFV declined in the introversion group, but not in the extraversion group. Additionally, an interaction between cerebral hemisphere and personality revealed that extraverts were recruiting resources from both the left and right cerebral hemispheres, while introverts only recruited resources from the right hemisphere. The results suggest that extraverts can allocate compensatory effort to mask performance differences. We discuss the theoretical and practical implications of these findings and offer future research directions that may help us understand these effects.

  1. Decoding regulatory landscape of somatic embryogenesis reveals differential regulatory networks between japonica and indica rice subspecies.

    PubMed

    Indoliya, Yuvraj; Tiwari, Poonam; Chauhan, Abhisekh Singh; Goel, Ridhi; Shri, Manju; Bag, Sumit Kumar; Chakrabarty, Debasis

    2016-03-14

    Somatic embryogenesis is a unique process in plants and has considerable interest for biotechnological application. Compare to japonica, indica rice has been less responsive to in vitro culture. We used Illumina Hiseq 2000 sequencing platform for comparative transcriptome analysis between two rice subspecies at six different developmental stages combined with a tag-based digital gene expression profiling. Global gene expression among different samples showed greater complexity in japonica rice compared to indica which may be due to polyphyletic origin of two rice subspecies. Expression pattern in initial stage indicate major differences in proembryogenic callus induction phase that may serve as key regulator to observe differences between both subspecies. Our data suggests that phytohormone signaling pathways consist of elaborate networks with frequent crosstalk, thereby allowing plants to regulate somatic embryogenesis pathway. However, this crosstalk varies between the two rice subspecies. Down regulation of positive regulators of meristem development (i.e. KNOX, OsARF5) and up regulation of its counterparts (OsRRs, MYB, GA20ox1/GA3ox2) in japonica may be responsible for its better regeneration and differentiation of somatic embryos. Comprehensive gene expression information in the present experiment may also facilitate to understand the monocot specific meristem regulation for dedifferentiation of somatic cell to embryogenic cells.

  2. Antibodies Raised Against Chlamydial Lipopolysaccharide Antigens Reveal Convergence in Germline Gene Usage and Differential Epitope Recognition

    PubMed Central

    Brooks, Cory L; Müller-Loennies, Sven; Borisova, Svetlana N.; Brade, Lore; Kosma, Paul; Hirama, Tomoko; MacKenzie, C. Roger; Brade, Helmut; Evans, Stephen V

    2011-01-01

    In order to explore monoclonal antibody recognition carbohydrate antigens, several structures from two monoclonal antibodies directed against carbohydrate epitopes derived from chlamydial LPS have been solved to high resolution. With the exception of CDR H3, antibodies S54-10 and S73-2 are both derived from the same set of germline gene segments as the previously reported structures S25-2 and S45-18. Despite this similarity, the antibodies differ in specificity and the mechanism by which they recognize their cognate antigen. S54-10 uses an unrelated CDR H3 to recognize its antigen in a fashion analogous to S45-18; however, S73-2 recognizes the same antigen as S45-18 and S54-10 in a wholly unrelated manner. Together, these antibody-antigen structures provide snapshots into how the immune system uses the same set of inherited germline gene segments to generate multiple possible specificities that allow for differential recognition of epitopes, and how unrelated CDR H3 sequences can result in convergent binding of clinically-relevant bacterial antigens. PMID:20000757

  3. Proteomic analysis reveals differentially expressed proteins in the rat frontal cortex after methamphetamine treatment.

    PubMed

    Faure, J J; Hattingh, S M; Stein, D J; Daniels, W M

    2009-12-01

    Methamphetamine (MA) is an addictive psycho-stimulant and the illicit use of the drug is escalating. In the present study, we examined protein expression profiles in the rat frontal cortex exposed to a total of eight MA injections (1 mg/kg, intraperitoneal) using 2-DE based proteomics. We investigated protein changes occurring in both the cytosolic fraction and the membrane fraction. 2-DE analysis resulted in 62 cytosolic and 44 membrane protein spots that were differentially regulated in the frontal cortex of rats exposed to MA when compared to control animals. Of these spots, 47 cytosolic and 42 membrane proteins were identified respectively, using ESI-Quad-TOF, which included ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), beta-synuclein, 78 kDa glucose-regulated protein (GRP 78), gamma-enolase, dihydropyrimidase-related protein 2 (DRP 2), complexin 2 and synapsin II. These proteins are associated with protein degradation, redox regulation, energy metabolism, cellular growth, cytoskeletal modifications and synaptic function. Proteomic research may be useful in exploring the complex underlying molecular mechanisms of MA dependence.

  4. Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes

    PubMed Central

    Hawley, Alyse K.; Brewer, Heather M.; Norbeck, Angela D.; Paša-Tolić, Ljiljana; Hallam, Steven J.

    2014-01-01

    Marine oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified waters. Currently OMZs are expanding due to global climate change with resulting feedback on marine ecosystem function. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally stratified fjord to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification, and inorganic carbon fixation were differentially expressed across the redoxcline and covaried with distribution patterns of ubiquitous OMZ microbes including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters, and denitrification, sulfur oxidation, and inorganic carbon fixation pathways affiliated with the SUP05 group of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic waters. Nitrifier nitrite oxidation and anammox pathways affiliated with Nirospina, Nitrospira, and Planctomycetes, respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The numerical abundance of SUP05 proteins mediating inorganic carbon fixation under anoxic conditions suggests that SUP05 will become increasingly important in global ocean carbon and nutrient cycling as OMZs expand. PMID:25053816

  5. Differential Proteomics of Urinary Exovesicles from Classical Galactosemic Patients Reveals Subclinical Kidney Insufficiency.

    PubMed

    Staubach, Simon; Pekmez, Murat; Hanisch, Franz-Georg

    2016-06-03

    Classical galactosemia is caused by a nearly complete deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), resulting in a severely impaired galactose metabolism with galactose-1-phosphate and galactitol accumulation. Even on a galactose-restricted diet, patients develop serious long-term complications of the central nervous system and ovaries that may result from chronic cell-toxic effects exerted by endogenous galactose. To address the question of whether disease-associated cellular perturbations could affect the kidney function of the patients, we performed differential proteomics of detergent-resistant membranes from urinary exovesicles. Galactosemic samples (showing drastic shifts from high-mannose to complex-type N-glycosylation on exosomal N-glycoproteins) and healthy, sex-matched controls were analyzed in quadruplex iTRAQ experiments performed in biological and technical replicates. Particularly in the female patient group, the most striking finding was a drastic increase of abundant serum (glyco)proteins, like albumin, leucine-rich α-2-glycoprotein, fetuin, immunoglobulins, prostaglandin H2 d-isomerase, and α-1-microglobulin protein (AMBP), pointing to a subclinical failure of kidney filter function in galactosemic patients and resulting in a heavy overload of exosomal membranes with adsorbed serum (glyco)proteins. Several of these proteins are connected to TBMN and IgAN, proteinuria, and renal damage. The impairment of renal protein filtration was also indicated by increased protein contents derived from extracellular matrices and lysosomes.

  6. Decoding regulatory landscape of somatic embryogenesis reveals differential regulatory networks between japonica and indica rice subspecies

    PubMed Central

    Indoliya, Yuvraj; Tiwari, Poonam; Chauhan, Abhisekh Singh; Goel, Ridhi; Shri, Manju; Bag, Sumit Kumar; Chakrabarty, Debasis

    2016-01-01

    Somatic embryogenesis is a unique process in plants and has considerable interest for biotechnological application. Compare to japonica, indica rice has been less responsive to in vitro culture. We used Illumina Hiseq 2000 sequencing platform for comparative transcriptome analysis between two rice subspecies at six different developmental stages combined with a tag-based digital gene expression profiling. Global gene expression among different samples showed greater complexity in japonica rice compared to indica which may be due to polyphyletic origin of two rice subspecies. Expression pattern in initial stage indicate major differences in proembryogenic callus induction phase that may serve as key regulator to observe differences between both subspecies. Our data suggests that phytohormone signaling pathways consist of elaborate networks with frequent crosstalk, thereby allowing plants to regulate somatic embryogenesis pathway. However, this crosstalk varies between the two rice subspecies. Down regulation of positive regulators of meristem development (i.e. KNOX, OsARF5) and up regulation of its counterparts (OsRRs, MYB, GA20ox1/GA3ox2) in japonica may be responsible for its better regeneration and differentiation of somatic embryos. Comprehensive gene expression information in the present experiment may also facilitate to understand the monocot specific meristem regulation for dedifferentiation of somatic cell to embryogenic cells. PMID:26973288

  7. Becoming Irreplaceable: How Comparisons to the Partner’s Alternatives Differentially Affect Low and High Self-Esteem People

    PubMed Central

    Murray, Sandra L.; Leder, Sadie; McClellan, Jennifer C. D.; Holmes, John G.; Pinkus, Rebecca T.; Harris, Brianna

    2009-01-01

    It is proposed that people are motivated to feel hard to replace in romantic relationships because feeling irreplaceable fosters trust in a partner’s continued responsiveness. By contrast, feeling replaceable motivates compensatory behavior aimed at strengthening the partner’s commitment to the relationship. A correlational study of dating couples and 2 experiments examined how satiating/thwarting the goal of feeling irreplaceable differentially affects relationship perception and behavior for low and high self-esteem people. The results revealed that satiating the goal of feeling irreplaceable increases trust for people low in self-esteem. In contrast, thwarting the goal of feeling irreplaceable increases compensatory behaviors meant to prove one’s indispensability for people high in self-esteem. PMID:20161401

  8. Expression Microarray Analysis Reveals Alternative Splicing of LAMA3 and DST Genes in Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Li, Ryan; Ochs, Michael F.; Ahn, Sun Mi; Hennessey, Patrick; Tan, Marietta; Soudry, Ethan; Gaykalova, Daria A.; Uemura, Mamoru; Brait, Mariana; Shao, Chunbo; Westra, William; Bishop, Justin; Fertig, Elana J.; Califano, Joseph A.

    2014-01-01

    Purpose Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors. Experimental Design We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort. Results After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001). Conclusion Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression. PMID:24675808

  9. Alternative exon usage creates novel transcript variants of tumor suppressor SHREW-1 gene with differential tissue expression profile

    PubMed Central

    Klemmt, Petra A. B.; Resch, Eduard; Smyrek, Isabell; Engels, Knut; Stelzer, Ernst H. K.

    2016-01-01

    ABSTRACT Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with β-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants. PMID:27870635

  10. Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

    PubMed Central

    Moreira-Leite, Flavia; Varga, Vladimir; Gull, Keith

    2016-01-01

    The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics. PMID:27519801

  11. Coseismic fault zone deformation revealed with differential lidar: Examples from Japanese Mw ∼7 intraplate earthquakes

    NASA Astrophysics Data System (ADS)

    Nissen, Edwin; Maruyama, Tadashi; Ramon Arrowsmith, J.; Elliott, John R.; Krishnan, Aravindhan K.; Oskin, Michael E.; Saripalli, Srikanth

    2014-11-01

    We use two recent Japanese earthquakes to demonstrate the rich potential, as well as some of the challenges, of differencing repeat airborne Light Detection and Ranging (lidar) topographic data to measure coseismic fault zone deformation. We focus on densely-vegetated sections of the 14 June 2008 Iwate-Miyagi (Mw 6.9) and 11 April 2011 Fukushima-Hamadori (Mw 7.1) earthquake ruptures, each covered by 2 m-resolution pre-event and 1 m-resolution post-event bare Earth digital terrain models (DTMs) obtained from commercial lidar providers. Three-dimensional displacements and rotations were extracted from these datasets using an adaptation of the Iterative Closest Point (ICP) algorithm. These displacements remain coherent close to surface fault breaks, as well as within dense forest, despite intervals of ∼2 years (Iwate-Miyagi) and ∼4 years (Fukushima-Hamadori) encompassed by the lidar scenes. Differential lidar analysis is thus complementary to Interferometric Synthetic Aperture Radar (InSAR) and sub-pixel correlation techniques which often break down under conditions of long time intervals, dense vegetation or steep displacement gradients. Although the ICP displacements are much noisier than overlapping InSAR line-of-sight displacements, they still provide powerful constraints on near-surface fault slip. In the Fukushima-Hamadori case, near-fault displacements and rotations are consistent with decreased primary fault slip at very shallow depths of a few tens of meters, helping to account for the large, along-strike heterogeneity in surface offsets observed in the field. This displacement field also captures long-wavelength deformation resulting from the 11 March 2011 Tohoku great earthquake.

  12. Suppression subtractive hybridization reveals differentially expressed genes in supraspinous ligaments of patients with ankylosing spondylitis.

    PubMed

    Zhang, Ying; Hu, Xu; Zhang, Chao; Zhou, Yue; Chu, Tong-Wei

    2015-06-01

    Ankylosing spondylitis (AS) is a severe chronic inflammatory disease that may ultimately result in the development of a 'bamboo‑like' spine. Although the pathological changes that occur in AS have been extensively investigated, the mechanism underlying spinal fusion during AS remains elusive. Differentially expressed genes (DEGs) in paraspinal tissues from patients with AS compared with those from healthy controls were therefore investigated. Polymerase chain reaction (PCR)‑based suppression subtractive hybridization was performed using total mRNA from the supraspinal ligaments of three patients with AS and three patients with spinal fractures as controls. From this, 27 genes were identified in all of the three independent forward libraries, which were defined as DEGs associated with AS. Reverse transcription‑quantitative PCR demonstrated that six DEGs were overexpressed in the tissues from patients with AS compared with those from individuals in the control group, including those encoding transforming growth factor β types I and III receptor, vascular endothelial growth factor, matrix metalloproteinase‑3, core‑binding factor α1 and bone morphogenetic protein 2. Western blot analysis showed increased expression in all six of these proteins in the samples from patients with AS compared with those in the control groups. These findings suggested that changes in the expression of these genes and proteins are associated with the development of spinal fusion during the pathogenesis of AS. Furthermore, these genes may be novel markers of the risk of developing AS, in addition to being targets for the treatment of this disease.

  13. [Differential expression of genes related to photoperiod-temperature sensitive genic male sterility in wheat, revealed by mRNA differential display using G-box family primer].

    PubMed

    Cao, Shuang-He; Liu, Dong-Cheng; Liu, Li-Ke; Guo, Xiao-Li; Zhang, Ai-Min

    2003-01-01

    mRNA differential display with G-box family primer was used to analyze the differential expression of genes of the photoperiod-temperature sensitive genic male sterile(PTSGMS) line of wheat, BAU3338, between the sterile and fertile conditions. The result indicated that gene expression was significantly different between the two types of condition during the fertility transformation phase. The twelve qualitatively different DNA bands were identified with reverse Northern blot hybridization and five positive clones, HT1-G10, HT1-G3, HT2-G2, HT1-G4 and HT2-G5 were sequenced. The homology search indicated that HT1-G10 was highly homological (96%) to the partial sequences of Triticum aestivum chloroplast genes, rbcL and atpB, HT1-G3 was also homological (88%) to Triticum aestivum histone H2A gene and the other three gene fragments were new sequences in Gen-Bank. The analysis of the candidate gene fragments supplied some effective evidences to reveal the developmental mechanism of PTSGMS.

  14. Amino-termini isoforms of the Slack K+ channel, regulated by alternative promoters, differentially modulate rhythmic firing and adaptation

    PubMed Central

    Brown, Maile R; Kronengold, Jack; Gazula, Valeswara-Rao; Spilianakis, Charalampos G; Flavell, Richard A; von Hehn, Christian A A; Bhattacharjee, Arin; Kaczmarek, Leonard K

    2008-01-01

    data suggest that alternative promoters of the Slack gene differentially modulate the properties of neurones. PMID:18787033

  15. RAP-PCR fingerprinting reveals time-dependent expression of development-related genes following differentiation process of Bacillus thuringiensis.

    PubMed

    Huang, Tianpei; Yu, Xiaomin; Gelbič, Ivan; Guan, Xiong

    2015-09-01

    Gene expression profiles are important data to reveal the functions of genes putatively involved in crucial biological processes. RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and specifically primed reverse transcription polymerase chain reaction (RT-PCR) were combined to screen differentially expressed genes following development of a commercial Bacillus thuringiensis subsp. kurstaki strain 8010 (serotype 3a3b). Six differentially expressed transcripts (RAP1 to RAP6) were obtained. RAP1 encoded a putative triple helix repeat-containing collagen or an exosporium protein H related to spore pathogenicity. RAP2 was homologous to a ClpX protease and an ATP-dependent protease La (LonB), which likely acted as virulence factors. RAP3 was homologous to a beta subunit of propionyl-CoA carboxylase required for the development of Myxococcus xanthus. RAP4 had homology to a quinone oxidoreductase involved in electron transport and ATP formation. RAP5 showed significant homology to a uridine kinase that mediates phosphorylation of uridine and azauridine. RAP6 shared high sequence identity with 3-methyl-2-oxobutanoate-hydroxymethyltransferase (also known as ketopantoate hydroxymethyltransferase or PanB) involved in the operation of the tricarboxylic acid cycle. The findings described here would help to elucidate the molecular mechanisms underlying the differentiation process of B. thuringiensis and unravel novel pathogenic genes.

  16. Cardioinductive network guiding stem cell differentiation revealed by proteomic cartography of tumor necrosis factor alpha-primed endodermal secretome.

    PubMed

    Arrell, D Kent; Niederländer, Nicolas J; Faustino, Randolph S; Behfar, Atta; Terzic, Andre

    2008-02-01

    In the developing embryo, instructive guidance from the ventral endoderm secures cardiac program induction within the anterolateral mesoderm. Endoderm-guided cardiogenesis, however, has yet to be resolved at the proteome level. Here, through cardiopoietic priming of the endoderm with the reprogramming cytokine tumor necrosis factor alpha (TNFalpha), candidate effectors of embryonic stem cell cardiac differentiation were delineated by comparative proteomics. Differential two-dimensional gel electrophoretic mapping revealed that more than 75% of protein species increased >1.5-fold in the TNFalpha-primed versus unprimed endodermal secretome. Protein spot identification by linear ion trap quadrupole (LTQ) tandem mass spectrometry (MS/MS) and validation by shotgun LTQ-Fourier transform MS/MS following multidimensional chromatography mapped 99 unique proteins from 153 spot assignments. A definitive set of 48 secretome proteins was deduced by iterative bioinformatic screening using algorithms for detection of canonical and noncanonical indices of secretion. Protein-protein interaction analysis, in conjunction with respective expression level changes, revealed a nonstochastic TNFalpha-centric secretome network with a scale-free hierarchical architecture. Cardiovascular development was the primary developmental function of the resolved TNFalpha-anchored network. Functional cooperativity of the derived cardioinductive network was validated through direct application of the TNFalpha-primed secretome on embryonic stem cells, potentiating cardiac commitment and sarcomerogenesis. Conversely, inhibition of primary network hubs negated the procardiogenic effects of TNFalpha priming. Thus, proteomic cartography establishes a systems biology framework for the endodermal secretome network guiding stem cell cardiopoiesis.

  17. Transcriptome analysis of neo-tetraploid rice reveals specific differential gene expressions associated with fertility and heterosis.

    PubMed

    Guo, Haibin; Mendrikahy, Jean Nestor; Xie, Lei; Deng, Junfeng; Lu, Zijun; Wu, Jinwen; Li, Xiang; Shahid, Muhammad Qasim; Liu, Xiangdong

    2017-01-10

    Polyploid rice hybrids have a powerful biological and yield potential that may become a new way for rice breeding; however, low fertility is major hindrance in commercial utilization. Here, we developed a neo-tetraploid rice that could overcome the sterility of autotetraploid rice and produce high heterosis. Transcriptome analysis of F1 hybrid developed by crossing neo-tetraploid with autotetraploid rice displayed 807, 663 and 866 differentially expressed genes that uniquely associated with F1 and specific to (DEGFu-sp) anther, ovary and leaf, respectively. Of the DEGFu-sp, 1224 genes displayed nonadditive expression; 44 and 10 genes were annotated as TFs and methyltransferase or hydroxymethyltransferase, respectively. Gene ontology enrichment and co-expression analysis revealed specific differential gene expressions in the DEGFu-sp to leaf, anther and ovary, such as genes related to photosynthesis, metabolic process and transport, and co-expression network including fertility, resistance and epigenetic elements. Of the DEGFu-sp to anther, 42 meiosis stage-specific genes, eight meiosis-related genes, such as RAD51 and SMC2, were identified. We identified 38 miRNAs from DEGFu-sp to anther, and their targets were associated with pollen fertility and retrotransposon protein. Our study provides new germplasm for polyploid rice breeding, and revealed complex regulatory mechanisms that might be associated with heterosis and fertility.

  18. Transcriptome analysis of neo-tetraploid rice reveals specific differential gene expressions associated with fertility and heterosis

    PubMed Central

    Guo, Haibin; Mendrikahy, Jean Nestor; Xie, Lei; Deng, Junfeng; Lu, Zijun; Wu, Jinwen; Li, Xiang; Shahid, Muhammad Qasim; Liu, Xiangdong

    2017-01-01

    Polyploid rice hybrids have a powerful biological and yield potential that may become a new way for rice breeding; however, low fertility is major hindrance in commercial utilization. Here, we developed a neo-tetraploid rice that could overcome the sterility of autotetraploid rice and produce high heterosis. Transcriptome analysis of F1 hybrid developed by crossing neo-tetraploid with autotetraploid rice displayed 807, 663 and 866 differentially expressed genes that uniquely associated with F1 and specific to (DEGFu-sp) anther, ovary and leaf, respectively. Of the DEGFu-sp, 1224 genes displayed nonadditive expression; 44 and 10 genes were annotated as TFs and methyltransferase or hydroxymethyltransferase, respectively. Gene ontology enrichment and co-expression analysis revealed specific differential gene expressions in the DEGFu-sp to leaf, anther and ovary, such as genes related to photosynthesis, metabolic process and transport, and co-expression network including fertility, resistance and epigenetic elements. Of the DEGFu-sp to anther, 42 meiosis stage-specific genes, eight meiosis-related genes, such as RAD51 and SMC2, were identified. We identified 38 miRNAs from DEGFu-sp to anther, and their targets were associated with pollen fertility and retrotransposon protein. Our study provides new germplasm for polyploid rice breeding, and revealed complex regulatory mechanisms that might be associated with heterosis and fertility. PMID:28071676

  19. Physiological and Transcriptional Analyses Reveal Differential Phytohormone Responses to Boron Deficiency in Brassica napus Genotypes

    PubMed Central

    Zhou, Ting; Hua, Yingpeng; Huang, Yupu; Ding, Guangda; Shi, Lei; Xu, Fangsen

    2016-01-01

    Phytohormones play pivotal roles in the response of plants to various biotic and abiotic stresses. Boron (B) is an essential microelement for plants, and Brassica napus (B. napus) is hypersensitive to B deficiency. However, how auxin responds to B deficiency remained a dilemma for many years and little is known about how other phytohormones respond to B deficiency. The identification of B-efficient/inefficient B. napus indicates that breeding might overcome these constraints in the agriculture production. Here, we seek to identify phytohormone-related processes underlying B-deficiency tolerance in B. napus at the physiological and gene expression levels. Our study indicated low-B reduced indole-3-acetic acid (IAA) concentration in both the shoots and roots of B. napus, and affected the expression of the auxin biosynthesis gene BnNIT1 and the efflux gene BnPIN1 in a time-dependent manner. Low-B increased the jasmonates (JAs) and abscisic acid (ABA) concentrations and induced the expression of the ABA biosynthesis gene BnNCED3 and the ABA sensor gene BnPYL4 in the shoot. In two contrasting genotypes, the auxin concentration decreased more drastically in the B-inefficient genotype ‘W10,’ and together the expression of BnNIT1 and BnPIN1 also decreased more significantly in ‘W10’ under long-term B deficiency. While the JAs concentration was considerably higher in this genotype, and the ABA concentration was induced in ‘W10’ compared with the B-efficient genotype ‘QY10.’ Digital gene expression (DGE) profiling confirmed the differential expression of the phytohormone-related genes, indicating more other phyohormone differences involving in gene regulation between ‘QY10’ and ‘W10’ under low-B stress. Additionally, the activity of DR5:GFP was reduced in the root under low-B in Arabidopsis, and the application of exogenous IAA could partly restore the B-defective phenotype in ‘W10.’ Overall, our data suggested that low-B disturbed phytohormone

  20. Differential effect of three base modifications on DNA thermostability revealed by high resolution melting.

    PubMed

    López, Carlos M Rodríguez; Lloyd, Amanda J; Leonard, Kate; Wilkinson, Mike J

    2012-09-04

    High resolution melting (HRM) can detect and quantify the presence of 5-methylcytosine (5mC) in DNA samples, but the ability of HRM to diagnose other DNA modifications remains unexplored. The DNA bases N6-methyladenine and 5-hydroxymethylcytosine occur across almost all phyla. While their function remains controversial, their presence perturbs DNA structure. Such modifications could affect gene regulation, chromatin condensation and DNA packaging. Here, we reveal that DNA containing N6-methyladenine or 5-hydroxymethylcytosine exhibits reduced thermal stability compared to cytosine-methylated DNA. These thermostability changes are sufficiently divergent to allow detection and quantification by HRM analysis. Thus, we report that HRM distinguishes between sequence-identical DNA differing only in the modification type of one base. This approach is also able to distinguish between two DNA fragments carrying both N6-methyladenine and 5-methylcytosine but differing only in the distance separating the modified bases. This finding provides scope for the development of new methods to characterize DNA chemically and to allow for low cost screening of mutant populations of genes involved in base modification. More fundamentally, contrast between the thermostabilizing effects of 5mC on dsDNA compared with the destabilizing effects of N6-methyladenine (m6A) and 5-hydroxymethylcytosine (5hmC) raises the intriguing possibility of an antagonistic relationship between modification types with functional significance.

  1. Genetic variation architecture of mitochondrial genome reveals the differentiation in Korean landrace and weedy rice.

    PubMed

    Tong, Wei; He, Qiang; Park, Yong-Jin

    2017-03-03

    Mitochondrial genome variations have been detected despite the overall conservation of this gene content, which has been valuable for plant population genetics and evolutionary studies. Here, we describe mitochondrial variation architecture and our performance of a phylogenetic dissection of Korean landrace and weedy rice. A total of 4,717 variations across the mitochondrial genome were identified adjunct with 10 wild rice. Genetic diversity assessment revealed that wild rice has higher nucleotide diversity than landrace and/or weedy, and landrace rice has higher diversity than weedy rice. Genetic distance was suggestive of a high level of breeding between landrace and weedy rice, and the landrace showing a closer association with wild rice than weedy rice. Population structure and principal component analyses showed no obvious difference in the genetic backgrounds of landrace and weedy rice in mitochondrial genome level. Phylogenetic, population split, and haplotype network evaluations were suggestive of independent origins of the indica and japonica varieties. The origin of weedy rice is supposed to be more likely from cultivated rice rather than from wild rice in mitochondrial genome level.

  2. Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation

    NASA Astrophysics Data System (ADS)

    Gentleman, Eileen; Swain, Robin J.; Evans, Nicholas D.; Boonrungsiman, Suwimon; Jell, Gavin; Ball, Michael D.; Shean, Tamaryn A. V.; Oyen, Michelle L.; Porter, Alexandra; Stevens, Molly M.

    2009-09-01

    An important aim of regenerative medicine is to restore tissue function with implantable, laboratory-grown constructs that contain tissue-specific cells that replicate the function of their counterparts in the healthy native tissue. It remains unclear, however, whether cells used in bone regeneration applications produce a material that mimics the structural and compositional complexity of native bone. By applying multivariate analysis techniques to micro-Raman spectra of mineralized nodules formed in vitro, we reveal cell-source-dependent differences in interactions between multiple bone-like mineral environments. Although osteoblasts and adult stem cells exhibited bone-specific biological activities and created a material with many of the hallmarks of native bone, the `bone nodules' formed from embryonic stem cells were an order of magnitude less stiff, and lacked the distinctive nanolevel architecture and complex biomolecular and mineral composition noted in the native tissue. Understanding the biological mechanisms of bone formation in vitro that contribute to cell-source-specific materials differences may facilitate the development of clinically successful engineered bone.

  3. Genetic variation architecture of mitochondrial genome reveals the differentiation in Korean landrace and weedy rice

    PubMed Central

    Tong, Wei; He, Qiang; Park, Yong-Jin

    2017-01-01

    Mitochondrial genome variations have been detected despite the overall conservation of this gene content, which has been valuable for plant population genetics and evolutionary studies. Here, we describe mitochondrial variation architecture and our performance of a phylogenetic dissection of Korean landrace and weedy rice. A total of 4,717 variations across the mitochondrial genome were identified adjunct with 10 wild rice. Genetic diversity assessment revealed that wild rice has higher nucleotide diversity than landrace and/or weedy, and landrace rice has higher diversity than weedy rice. Genetic distance was suggestive of a high level of breeding between landrace and weedy rice, and the landrace showing a closer association with wild rice than weedy rice. Population structure and principal component analyses showed no obvious difference in the genetic backgrounds of landrace and weedy rice in mitochondrial genome level. Phylogenetic, population split, and haplotype network evaluations were suggestive of independent origins of the indica and japonica varieties. The origin of weedy rice is supposed to be more likely from cultivated rice rather than from wild rice in mitochondrial genome level. PMID:28256554

  4. Thermodynamics imprinting reveals differential binding of metals to {alpha}-synuclein: Relevance to parkinson's disease

    SciTech Connect

    Bharathi; Rao, K.S.J. . E-mail: kjr5n@yahoo.co.in

    2007-07-20

    The aggregation of {alpha}-synuclein is a hallmark feature of Parkinson's disease (PD) and other synucleinopathies. Metals are the significant etiological factors in PD, and their interaction with {alpha}-synuclein affect dramatically the kinetics of fibrillation in vitro and are proposed to play an important and potential neurodegenerative role in vivo. In the present study, we investigated the stoichiometry of binding of copper [Cu (II)] and iron [Fe (III)] with {alpha}-synuclein (wild recombinant type and A30P, A53T, E46K mutant forms) using isothermal titration calorimetry (ITC). {alpha}-Synuclein monomer (wild and mutant forms) titrated by Cu (II), showed two binding sites, with an apparent K {sub B} of 10{sup 5} M and 10{sup 4} M, respectively. But, {alpha}-synuclein (wild type and mutant forms) titrated with Fe (III) revealed a K {sub B} of 10{sup 5} M with single binding site. The present investigation uncovers the detailed binding propensities between metals and {alpha}-synuclein and has biological implications in PD.

  5. Ability or Access-Ability: Differential Item Functioning of Items on Alternate Performance-Based Assessment Tests for Students with Visual Impairments

    ERIC Educational Resources Information Center

    Zebehazy, Kim T.; Zigmond, Naomi; Zimmerman, George J.

    2012-01-01

    Introduction: This study investigated differential item functioning (DIF) of test items on Pennsylvania's Alternate System of Assessment (PASA) for students with visual impairments and severe cognitive disabilities and what the reasons for the differences may be. Methods: The Wilcoxon signed ranks test was used to analyze differences in the scores…

  6. Differential proteomic analysis of an engineered Streptomyces coelicolor strain reveals metabolic pathways supporting growth on n-hexadecane.

    PubMed

    Gallo, Giuseppe; Lo Piccolo, Luca; Renzone, Giovanni; La Rosa, Ruggero; Scaloni, Andrea; Quatrini, Paola; Puglia, Anna Maria

    2012-06-01

    The alkB gene, encoding an alkane monooxygenase in the actinomycete Gordonia sp. SoCg, was expressed in the non-alkane-degrading actinomycete Streptomyces coelicolor M145. The resulting engineered strain, M145-AH, can grow on n-hexadecane as sole carbon source. To unravel proteins associated with growth on n-alkanes, proteome of M145-AH after 6, 24, and 48 h of incubation in the Bushnell-Haas (BH) mineral medium containing n-hexadecane as sole carbon source (H condition) and in BH without any carbon source (0 condition) were compared using 2D-differential gel electrophoresis. Proteome analysis revealed significant changes only at 48 h, showing 48 differentially abundant proteins identified by mass spectrometry procedures. To asses if these proteins were specifically related to n-hexadecane metabolism, their expression was investigated, comparing H proteome with that of M145-AH incubated in BH with glucose as sole carbon source (G condition). Thus, protein expression profiles at 6, 24, and 48 h under H, 0, and G conditions were combined, revealing that M145-AH regulates in a temporally- and carbon source-dependent manner the expression of proteins involved in regulatory events, central carbon metabolism, respiration, β-oxidation, membrane transport, and amino acid and protein metabolism. Interestingly, 21 % of them, mostly involved in membrane transport and protein metabolism, showed a n-hexadecane-dependent regulation with regulatory proteins such as CRP likely to have a key role in M145-AH n-hexadecane growth. These results, expanding the knowledge on n-alkane utilization in Gram-positive bacteria, reveal genes to be targeted to develop an efficient S. coelicolor M145-AH-based bioremediation system.

  7. Whole Hillslope Irrigation Reveals Differential Interflow Behavior of Dye Tracers, Conservative Solutes and Nutrients

    NASA Astrophysics Data System (ADS)

    Jackson, C. R.; Du, E.; Klaus, J.; Griffiths, N. A.; McDonnell, J. J.; Blake, J. I.

    2012-12-01

    Previous investigations of perching and interflow behavior in low angle hillslopes in the SC Coastal Plain have suggested a high threshold for interflow occurrence. Here we report a new irrigation experiment designed to quantify interflow thresholds and reveal subsurface mixing processes during steady state flow conditions over a 12m x 16.5m plot draining to an interflow interception trench. Dye tracers were applied on surface transects prior to irrigation, and bromide (conservative tracer), nitrate, ammonium, and phosphorus (reactive tracers) were added at constant concentrations to the irrigation water drawn from a deep aquifer with a distinct isotopic signature. 417mm of water were applied over 51 hours, and drainage conditions were monitored for a week following irrigation. Interflow in the two drains commenced after 131 and 178mm, and flow rates diminished immediately after irrigation ceased, although interflow continued for four more days. Over the experiment, 199mm of water (49% of applied water) appeared as interflow. Dye tracers moved rapidly with the wetting front, with peak concentrations measured shortly after flow commencement, suggesting saturated topsoil conductivities of 0.5 to 1.5 m/hr. No preferential flow was observed during this experiment or previously during rainfall events at the trench face. Bromide concentrations and the new water fraction rose steadily throughout irrigation, peaking about 16 hours after irrigation ceased. Ammonium and phosphorus concentrations at the trench face were low, suggesting rapid uptake or sorption, while nitrate concentrations were higher, suggesting more conservative transport. Our two collection drains showed identical temporal variation in bromide concentrations but consistently different new/old water fractions, indicating differences in flow paths and storages within the plot. These data suggest that tightly bound soil water exchanged with new water throughout the experiment, and that a significant portion

  8. Feather isotope analysis reveals differential patterns of habitat and resource use in populations of white-winged doves

    USGS Publications Warehouse

    Carleton, Scott A.; Martinez Del Rio, Carlos; Robinson, Timothy J.

    2015-01-01

    The white-winged dove (Zenaida asiatica) serves an important ecological role as a diurnal pollinator of the saguaro cactus in the Sonoran desert and an economic role as a highly sought after game bird in North America. White-winged doves are intimately linked to anthropogenic changes on the landscape and because of this, have experienced dramatic population fluctuations over the last 75 years in response, both positively and negatively, to anthropogenic changes on the landscape. To understand the factors driving population growth and decline of migratory species like the white-winged dove, it is imperative we study resource use on both their breeding and wintering grounds. To understand how populations are distributed on the wintering grounds, we tested an alternative to band recovery approaches by using stable isotope analysis. Before we could use isotope analysis to link breeding and wintering locations for this species, we first needed to determine if hydrogen (δ2H) and carbon (δ13C) stable isotopes in feather tissue (δ2Hf and δ13Cf, respectively) could differentiate among populations of white-winged doves across their breeding range in Texas, New Mexico, and Arizona. δ2Hf and δ13Cf not only differentiated between populations of white-winged doves that breed in the United States, but δ2Hf also provided further differentiation in white-winged doves that breed in native Sonoran Desert and agricultural habitats in the western portion of their range. Ecological processes associated with desert resources and anthropogenic influences, specifically saguaro cacti and irrigated crops, largely determined δ2Hf in some white-winged doves in Arizona whereas δ2H of precipitation (δ2Hp) largely determined δ2Hfof doves in New Mexico and Texas. This study highlights the usefulness of stable isotope analysis to differentiate populations of animals across the landscape and the insight isotopes can provide into habitat and resource use. Published 2015. This article

  9. Chitin synthase genes in Manduca sexta: characterization of a gut-specific transcript and differential tissue expression of alternately spliced mRNAs during development.

    PubMed

    Hogenkamp, David G; Arakane, Yasuyuki; Zimoch, Lars; Merzendorfer, Hans; Kramer, Karl J; Beeman, Richard W; Kanost, Michael R; Specht, Charles A; Muthukrishnan, Subbaratnam

    2005-06-01

    Chitin, the linear homopolymer of beta-1,4-linked N-acetylglucosamine, is produced by the enzyme chitin synthase (CHS). In general, this insoluble polysaccharide is found in two major extracellular structures in insects, the cuticle that overlays the epidermis and the peritrophic membrane (PM) that lines the midgut. Based on amino acid sequence similarities, insect CHSs are divided into two classes, A and B, and to date no more than two CHS genes have been identified in any single insect species. In species where both CHSs have been identified, one class A CHS and one class B CHS are always present. This finding suggests that these two genes may encode enzymes that synthesize chitin in different epithelial tissues. In our laboratory, we previously characterized transcripts for a class A CHS gene (MsCHS1) from the tobacco hornworm, Manduca sexta. We observed the expression of this gene in the larval epidermis, suggesting that the encoded enzyme functions to synthesize cuticular chitin. In this paper, we characterize a second chitin synthase gene (MsCHS2) belonging to class B and its cDNA from Manduca and show that it is expressed only in the midgut. This cDNA contains an open reading frame of 4575 nucleotides, which encodes a conceptual protein that is 1524 amino acids in length and is predicted to contain 16 transmembrane spans. Northern blot analysis of RNA isolated from anterior, medial, and posterior sections of the midgut from feeding larvae indicate that MsCHS2 is primarily expressed in the anterior midgut, with transcript levels tapering off in the medial and posterior midgut. Analysis of the MsCHS2 gene sequence indicates the absence of an alternate exon in contrast to the MsCHS1 gene, which yields two transcripts, MsCHS1a and MsCHS1b. RT-PCR analysis of the differential expression of these alternately spliced transcripts reveals that both splice variants are present in the epidermis. However, the ratio of the two alternately spliced transcripts varies

  10. RNA-sequencing analysis reveals the hepatotoxic mechanism of perfluoroalkyl alternatives, HFPO2 and HFPO4, following exposure in mice.

    PubMed

    Wang, Jianshe; Wang, Xiaoyang; Sheng, Nan; Zhou, Xiujuan; Cui, Ruina; Zhang, Hongxia; Dai, Jiayin

    2017-04-01

    The toxicological impact of traditional perfluoroalkyl chemicals has led to the elimination and restriction of these substances. However, many novel perfluoroalkyl alternatives remain unregulated and little is known about their potential effects on environmental and human health. Daily administration of two alternative perfluoroalkyl substances, HFPO2 and HFPO4 (1 mg kg(-1) body weight), for 28 days resulted in hepatomegaly and hepatic histopathological injury in mice, particularly in the HFPO4 group. We generated and compared high-throughput RNA-sequencing data from hepatic tissues in control and treatment group mice to clarify the mechanism of HFPO2 and HFPO4 hepatotoxicity. We identified 146 (101 upregulated, 45 downregulated) and 1295 (716 upregulated, 579 downregulated) hepatic transcripts that exhibited statistically significant changes (fold change ≥2 or ≤0.5, false discovery rate < 0.05) after HFPO2 and HFPO4 treatment, respectively. Among them, 111 (82 upregulated, 29 downregulated) transcripts were changed in both groups, and lipid metabolism associated genes were dominant. Thus, similar to their popular predecessors, HFPO2 and HFPO4 exposure exerted hepatic effects, including hepatomegaly and injury, and altered lipid metabolism gene levels in the liver, though HFPO4 exerted greater hepatotoxicity than HFPO2. The unregulated use of these emerging perfluoroalkyl alternatives may affect environmental and human health, and their biological effects need further exploration. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Compartmentalized gene expression profiling of receptive endometrium reveals progesterone regulated ENPP3 is differentially expressed and secreted in glycosylated form

    PubMed Central

    Boggavarapu, Nageswara Rao; Lalitkumar, Sujata; Joshua, Vijay; Kasvandik, Sergo; Salumets, Andres; Lalitkumar, Parameswaran Grace; Gemzell-Danielsson, Kristina

    2016-01-01

    The complexity of endometrial receptivity at the molecular level needs to be explored in detail to improve the management of infertility. Here, differential expression of transcriptomes in receptive endometrial glands and stroma revealed Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a progesterone regulated factor and confirmed by various methods, both at mRNA and protein level. The involvement of ENPP3 in embryo attachment was tested in an in vitro model for human embryo implantation. Interestingly, there was high expression of ENPP3 mRNA in stroma but not protein. Presence of N-glycosylated ENPP3 in receptive phase uterine fluid in women confirms its regulation by progesterone and makes it possible to use in a non-invasive test of endometrial receptivity. PMID:27665743

  12. Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis.

    PubMed

    Kayser, Manfred; Lao, Oscar; Anslinger, Katja; Augustin, Christa; Bargel, Grazyna; Edelmann, Jeanett; Elias, Sahar; Heinrich, Marielle; Henke, Jürgen; Henke, Lotte; Hohoff, Carsten; Illing, Anett; Jonkisz, Anna; Kuzniar, Piotr; Lebioda, Arleta; Lessig, Rüdiger; Lewicki, Slawomir; Maciejewska, Agnieszka; Monies, Dorota Marta; Pawłowski, Ryszard; Poetsch, Micaela; Schmid, Dagmar; Schmidt, Ulrike; Schneider, Peter M; Stradmann-Bellinghausen, Beate; Szibor, Reinhard; Wegener, Rudolf; Wozniak, Marcin; Zoledziewska, Magdalena; Roewer, Lutz; Dobosz, Tadeusz; Ploski, Rafal

    2005-09-01

    To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n = 913) and 11 from Germany (n = 1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r = 0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier's algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.

  13. Thermal stability and molecular microstructure of heat-induced cereal grains, revealed with Raman molecular microspectroscopy and differential scanning calorimetry.

    PubMed

    Khan, Md Majibur Rahman; Yu, Peiqiang

    2013-07-03

    The objectives of the present study were to use Raman molecular microspectroscopy and differential scanning calorimetry (DSC) to reveal molecular thermal stability and thermal degradation behavior of heat-induced cereal grains and reveal the molecular chemistry of the protein structures of cereal grain tissues affected by heat processing and to quantify the protein secondary structures using multicomponent peak modeling Gaussian and Lorentzian methods. Hierarchical cluster analysis (CLA) and principal components analysis (PCA) were also conducted to identify molecular differences in the Raman spectra. Three cereal grain seeds, wheat, triticale, and corn, were used as the model for feed protein in the experiment. The specimens were autoclaved (moist heating) and dry-heated (roasted) at 121 °C for 80 min, respectively. Raman spectroscopy results revealed that there are marked differences in the secondary structures of the proteins subjected to various heating treatments of different cereals. The sensitivity of cereals to moist heating was much higher than the sensitivity to dry heating. The multivariate analyses (CLA and PCA) showed that heat treatment was significantly isolated between the different Raman raw spectra. The DSC study revealed that the thermal degradation behavior of cereals was significantly changed after moist- and dry-heat treatments. The position of the major endothermic peak of dry-heated cereals shifted toward a higher temperature, from 131.7 to 134.0 °C, suggesting the high thermal stability of dry-heated cereals. In contrast, the endothermic peak position was slightly decreased to 132.1 °C in the case of moist autoclaved heating. The digestive behavior and nutritive value of rumen-undegradable protein in animals may be related to the changes of the protein secondary molecular structure and thermal stability of the cereal grain materials, which is attributed by Raman microspectroscopy and DSC endotherm profiles.

  14. Differential introgression reveals candidate genes for selection across a spruce (Picea sitchensis × P. glauca) hybrid zone.

    PubMed

    Hamilton, Jill A; Lexer, Christian; Aitken, Sally N

    2013-02-01

    Differential patterns of introgression between species across ecological gradients provide a fine-scale depiction of extrinsic and intrinsic factors that contribute to the maintenance of species barriers and adaptation across heterogeneous environments. Introgression was examined for 721 individuals collected from the ecological transition zone spanning maritime to continental climates within the Picea sitchensis-Picea glauca contact zone using a panel of 268 candidate gene single nucleotide polymorphisms. Geographic clines showed a strong spatial relationship between allele frequencies and both distance from the ocean along major rivers and mean annual precipitation, indicating a strong role for environmental selection. Interspecific patterns of differentiation using outlier tests revealed three candidate genes that may be targets of long-term divergent selection between the parental species, although contemporary genomic clines within the hybrid zone suggested neutral patterns of introgression for these genes. This study provides a fine-scale analysis of locus-specific introgression, identifying a suite of candidate loci that may be targets of extrinsic or intrinsic selection, with broad application in understanding local adaptation to climate.

  15. Microarray and Degradome Sequencing Reveal MicroRNA Differential Expression Profiles and Their Targets in Pinellia pedatisecta

    PubMed Central

    Lang, Qiulei; Zhou, Wei; Xu, Shaowei; Xu, Tao

    2013-01-01

    MicroRNAs (miRNAs) are endogenous small non-coding RNAs which play a critical role in gene regulation in plants. Pinelliapedatisecta is one of the most important herbs in traditional Chinese medicine, but there are no microRNAs of Pinelliapedatisecta were deposited in miRBase and the research of the related miRNA biological functions is still insufficient. To detect Pinelliapedatisecta miRNAs and discover their expression difference with Pinelliaternata, we carried out a microarray profiling. A total of 101 miRNAs belonging to 22 miRNA families were detected both in Pinelliapedatisecta and Pinelliaternata respectively, among them 21 miRNAs showed their differentially expression. GO (gene ontology) term enrichment analysis of the target genes of differential expression miRNAs reveal that these miRNAs mainly affect the reproduction, transcription factor activity and plant developmental process. To elucidate the target function of miRNAs, we constructed a degradome library from Pinellia pedatisecta leaf. The result showed that a total of 18 transcript were identified as targets of miRNAs and further analysis indicated that miR156 and miR529 may function together to repress SPL14. PMID:24086673

  16. Negative differential mobility for negative carriers as revealed by space charge measurements on crosslinked polyethylene insulated model cables

    NASA Astrophysics Data System (ADS)

    Teyssedre, G.; Vu, T. T. N.; Laurent, C.

    2015-12-01

    Among features observed in polyethylene materials under relatively high field, space charge packets, consisting in a pulse of net charge that remains in the form of a pulse as it crosses the insulation, are repeatedly observed but without complete theory explaining their formation and propagation. Positive charge packets are more often reported, and the models based on negative differential mobility(NDM) for the transport of holes could account for some charge packets phenomenology. Conversely, NDM for electrons transport has never been reported so far. The present contribution reports space charge measurements by pulsed electroacoustic method on miniature cables that are model of HVDC cables. The measurements were realized at room temperature or with a temperature gradient of 10 °C through the insulation under DC fields on the order 30-60 kV/mm. Space charge results reveal systematic occurrence of a negative front of charges generated at the inner electrode that moves toward the outer electrode at the beginning of the polarization step. It is observed that the transit time of the front of negative charge increases, and therefore the mobility decreases, with the applied voltage. Further, the estimated mobility, in the range 10-14-10-13 m2 V-1 s-1 for the present results, increases when the temperature increases for the same condition of applied voltage. The features substantiate the hypothesis of negative differential mobility used for modelling space charge packets.

  17. Gait Analysis at Multiple Speeds Reveals Differential Functional and Structural Outcomes in Response to Graded Spinal Cord Injury

    PubMed Central

    Krizsan-Agbas, Dora; Winter, Michelle K.; Eggimann, Linda S.; Meriwether, Judith; Berman, Nancy E.; McCarson, Kenneth E.

    2014-01-01

    Abstract Open-field behavioral scoring is widely used to assess spinal cord injury (SCI) outcomes, but has limited usefulness in describing subtle changes important for posture and locomotion. Additional quantitative methods are needed to increase the resolution of locomotor outcome assessment. This study used gait analysis at multiple speeds (GAMS) across a range of mild-to-severe intensities of thoracic SCI in the rat. Overall, Basso, Beattie, and Bresnahan (BBB) scores and subscores were assessed, and detailed automated gait analysis was performed at three fixed walking speeds (3.5, 6.0, and 8.5 cm/sec). Variability in hindpaw brake, propel, and stance times were analyzed further by integrating across the stance phase of stepping cycles. Myelin staining of spinal cord sections was used to quantify white matter loss at the injury site. Varied SCI intensity produced graded deficits in BBB score, BBB subscores, and spinal cord white matter and total volume loss. GAMS measures of posture revealed decreased paw area, increased limb extension, altered stance width, and decreased values for integrated brake, propel, and stance. Measures of coordination revealed increased stride frequency concomitant with decreased stride length, resulting in deviation from consistent forelimb/hindlimb coordination. Alterations in posture and coordination were correlated to impact severity. GAMS results correlated highly with functional and histological measures and revealed differential relationships between sets of GAMS dynamics and cord total volume loss versus epicenter myelin loss. Automated gait analysis at multiple speeds is therefore a useful tool for quantifying nuanced changes in gait as an extension of histological and observational methods in assessing SCI outcomes. PMID:24405378

  18. Gait analysis at multiple speeds reveals differential functional and structural outcomes in response to graded spinal cord injury.

    PubMed

    Krizsan-Agbas, Dora; Winter, Michelle K; Eggimann, Linda S; Meriwether, Judith; Berman, Nancy E; Smith, Peter G; McCarson, Kenneth E

    2014-05-01

    Open-field behavioral scoring is widely used to assess spinal cord injury (SCI) outcomes, but has limited usefulness in describing subtle changes important for posture and locomotion. Additional quantitative methods are needed to increase the resolution of locomotor outcome assessment. This study used gait analysis at multiple speeds (GAMS) across a range of mild-to-severe intensities of thoracic SCI in the rat. Overall, Basso, Beattie, and Bresnahan (BBB) scores and subscores were assessed, and detailed automated gait analysis was performed at three fixed walking speeds (3.5, 6.0, and 8.5 cm/sec). Variability in hindpaw brake, propel, and stance times were analyzed further by integrating across the stance phase of stepping cycles. Myelin staining of spinal cord sections was used to quantify white matter loss at the injury site. Varied SCI intensity produced graded deficits in BBB score, BBB subscores, and spinal cord white matter and total volume loss. GAMS measures of posture revealed decreased paw area, increased limb extension, altered stance width, and decreased values for integrated brake, propel, and stance. Measures of coordination revealed increased stride frequency concomitant with decreased stride length, resulting in deviation from consistent forelimb/hindlimb coordination. Alterations in posture and coordination were correlated to impact severity. GAMS results correlated highly with functional and histological measures and revealed differential relationships between sets of GAMS dynamics and cord total volume loss versus epicenter myelin loss. Automated gait analysis at multiple speeds is therefore a useful tool for quantifying nuanced changes in gait as an extension of histological and observational methods in assessing SCI outcomes.

  19. A Method of Accounting for Enzyme Costs in Flux Balance Analysis Reveals Alternative Pathways and Metabolite Stores in an Illuminated Arabidopsis Leaf.

    PubMed

    Cheung, C Y Maurice; Ratcliffe, R George; Sweetlove, Lee J

    2015-11-01

    Flux balance analysis of plant metabolism is an established method for predicting metabolic flux phenotypes and for exploring the way in which the plant metabolic network delivers specific outcomes in different cell types, tissues, and temporal phases. A recurring theme is the need to explore the flexibility of the network in meeting its objectives and, in particular, to establish the extent to which alternative pathways can contribute to achieving specific outcomes. Unfortunately, predictions from conventional flux balance analysis minimize the simultaneous operation of alternative pathways, but by introducing flux-weighting factors to allow for the variable intrinsic cost of supporting each flux, it is possible to activate different pathways in individual simulations and, thus, to explore alternative pathways by averaging thousands of simulations. This new method has been applied to a diel genome-scale model of Arabidopsis (Arabidopsis thaliana) leaf metabolism to explore the flexibility of the network in meeting the metabolic requirements of the leaf in the light. This identified alternative flux modes in the Calvin-Benson cycle revealed the potential for alternative transitory carbon stores in leaves and led to predictions about the light-dependent contribution of alternative electron flow pathways and futile cycles in energy rebalancing. Notable features of the analysis include the light-dependent tradeoff between the use of carbohydrates and four-carbon organic acids as transitory storage forms and the way in which multiple pathways for the consumption of ATP and NADPH can contribute to the balancing of the requirements of photosynthetic metabolism with the energy available from photon capture.

  20. A Method of Accounting for Enzyme Costs in Flux Balance Analysis Reveals Alternative Pathways and Metabolite Stores in an Illuminated Arabidopsis Leaf1[OPEN

    PubMed Central

    Cheung, C.Y. Maurice; Ratcliffe, R. George; Sweetlove, Lee J.

    2015-01-01

    Flux balance analysis of plant metabolism is an established method for predicting metabolic flux phenotypes and for exploring the way in which the plant metabolic network delivers specific outcomes in different cell types, tissues, and temporal phases. A recurring theme is the need to explore the flexibility of the network in meeting its objectives and, in particular, to establish the extent to which alternative pathways can contribute to achieving specific outcomes. Unfortunately, predictions from conventional flux balance analysis minimize the simultaneous operation of alternative pathways, but by introducing flux-weighting factors to allow for the variable intrinsic cost of supporting each flux, it is possible to activate different pathways in individual simulations and, thus, to explore alternative pathways by averaging thousands of simulations. This new method has been applied to a diel genome-scale model of Arabidopsis (Arabidopsis thaliana) leaf metabolism to explore the flexibility of the network in meeting the metabolic requirements of the leaf in the light. This identified alternative flux modes in the Calvin-Benson cycle revealed the potential for alternative transitory carbon stores in leaves and led to predictions about the light-dependent contribution of alternative electron flow pathways and futile cycles in energy rebalancing. Notable features of the analysis include the light-dependent tradeoff between the use of carbohydrates and four-carbon organic acids as transitory storage forms and the way in which multiple pathways for the consumption of ATP and NADPH can contribute to the balancing of the requirements of photosynthetic metabolism with the energy available from photon capture. PMID:26265776

  1. Serial analysis of gene expression in sugarcane (Saccharum spp.) leaves revealed alternative C4 metabolism and putative antisense transcripts.

    PubMed

    Calsa, Tercilio; Figueira, Antonio

    2007-04-01

    Sugarcane (Saccharum spp.) is a highly efficient biomass and sugar producing crop. Leaf reactions have been considered as potential rate-limiting step for sucrose accumulation in sugarcane stalks. To characterize the sugarcane leaf transcriptome, field-grown mature leaves from cultivar "SP80-3280" were analyzed using Serial Analysis of Gene Expression (SAGE). From 480 sequenced clones, 9,482 valid tags were extracted, with 5,227 unique sequences, from which 3,659 (70%) matched at least a sugarcane assembled sequence (SAS) with putative function; while 872 tags (16.7%) matched SAS with unknown function; 523 (10%) matched SAS without a putative annotation; and only 173 (3.3%) did not match any sugarcane ESTs. Based on gene ontology (GO), photosystem (PS) I reaction center was identified as the most frequent gene product location, followed by the remaining sites of PS I, PS II and thylakoid complexes. For metabolic processes, photosynthesis light harvesting complexes; carbon fixation; and chlorophyll biosynthesis were the most enriched GO-terms. Considering the alternative photosynthetic C(4) cycles, tag frequencies related to phosphoenolpyruvate carboxykinase (PEPCK) and aspartate aminotransferase compared to those for NADP(+)-malic enzyme (NADP-ME) and NADP-malate dehydrogenase, suggested that PEPCK-type decarboxylation appeared to predominate over NADP-ME in mature leaves, although both may occur, opposite to currently assumed in sugarcane. From the unique tag set, 894 tags (17.1%) were assigned as potentially derived from antisense transcripts, while 73 tags (1.4%) were assigned to more than one SAS, suggesting the occurrence of alternative processing. The occurrence of antisense was validated by quantitative reverse transcription amplification. Sugarcane leaf transcriptome provided new insights for functional studies associated with sucrose synthesis and accumulation.

  2. High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression.

    PubMed

    Batut, Philippe; Dobin, Alexander; Plessy, Charles; Carninci, Piero; Gingeras, Thomas R

    2013-01-01

    Many eukaryotic genes possess multiple alternative promoters with distinct expression specificities. Therefore, comprehensively annotating promoters and deciphering their individual regulatory dynamics is critical for gene expression profiling applications and for our understanding of regulatory complexity. We introduce RAMPAGE, a novel promoter activity profiling approach that combines extremely specific 5'-complete cDNA sequencing with an integrated data analysis workflow, to address the limitations of current techniques. RAMPAGE features a streamlined protocol for fast and easy generation of highly multiplexed sequencing libraries, offers very high transcription start site specificity, generates accurate and reproducible promoter expression measurements, and yields extensive transcript connectivity information through paired-end cDNA sequencing. We used RAMPAGE in a genome-wide study of promoter activity throughout 36 stages of the life cycle of Drosophila melanogaster, and describe here a comprehensive data set that represents the first available developmental time-course of promoter usage. We found that >40% of developmentally expressed genes have at least two promoters and that alternative promoters generally implement distinct regulatory programs. Transposable elements, long proposed to play a central role in the evolution of their host genomes through their ability to regulate gene expression, contribute at least 1300 promoters shaping the developmental transcriptome of D. melanogaster. Hundreds of these promoters drive the expression of annotated genes, and transposons often impart their own expression specificity upon the genes they regulate. These observations provide support for the theory that transposons may drive regulatory innovation through the distribution of stereotyped cis-regulatory modules throughout their host genomes.

  3. X-ray Crystallographic Studies Reveal That the Incorporation of Spacer Groups in Carbonic Anhydrase Inhibitors Causes Alternate Binding Modes

    SciTech Connect

    Fisher,S.; Govindasamy, L.; Boyle, N.; Agbandje-McKenna, M.; Silverman, D.; Blackburn, G.; McKenna, R.

    2006-01-01

    Human carbonic anhydrases (CAs) are well studied targets for the development of inhibitors for pharmaceutical applications. The crystal structure of human CA II has been determined in complex with two CA inhibitors (CAIs) containing conventional sulfonamide and thiadiazole moieties separated by a -CF{sub 2}- or -CHNH{sub 2}- spacer group. The structures presented here reveal that these spacer groups allow novel binding modes for the thiadiazole moiety compared with conventional CAIs.

  4. Id-1B, an alternatively spliced isoform of the inhibitor of differentiation-1, impairs cancer cell malignancy through inhibition of proliferation and angiogenesis.

    PubMed

    Nguewa, P; Manrique, I; Díaz, R; Redrado, M; Parrondo, R; Perez-Stable, C; Calvo, A

    2014-01-01

    Id-1 is a member of the helix-loop-helix family of proteins that regulates the activity of transcription factors to suppress cellular differentiation and to promote cell growth. Overexpression of Id-1 in tumor cells correlates with increased malignancy and resistance to chemotherapy and radiotherapy. Id-1B is an isoform generated by alternative splicing that differs from the classical Id-1 in the 13-C-terminal amino acids, whose function is at present unknown. We have studied the role of Id-1B in cancer and its expression in healthy/malignant lung tissues. Overexpression of Id-1B in A549 lung and PC3 prostate cancer cells reduced anchorage-dependent and independent proliferation and clonogenic potential. Moreover, it increased the proportion of cells in the G0/G1 phase of the cell cycle and p27 levels, while reduced phospho-Erk and cyclin A levels. Through microarray analysis, we identified genes involved in cell growth and proliferation that are specifically deregulated as a consequence of Id-1B overexpression, including IGF2, BMP4, Id2, GATA3, EREG and AREG. Id-1B overexpressing cells that were treated with 4Gy irradiation dose were significantly less resistant to cell death. In vivo assays demonstrated that tumors with high Id-1B levels exhibited less growth (p<0.01), metabolic activity (glucose uptake) and angiogenesis (p<0.05) compared to tumors with low Id-1B expression; mice survival was significantly extended (p<0.05). Quantification by qRT-PCR revealed that expression of Id-1B was significantly lower (p<0.01) in human lung tumors compared to their matched nonmalignant counterparts. In conclusion, our results demonstrate that Id-1B decreases the malignancy of lung and prostate cancer cells, sensitizes them to radiotherapy-induced cell death, and counteracts the protumorigenic role of the classical form of Id-1.

  5. Differential Replication of Two Chloroplast Genome Forms in Heteroplasmic Chlamydomonas reinhardtii Gametes Contributes to Alternative Inheritance Patterns

    PubMed Central

    Nishimura, Yoshiki; Stern, David B.

    2010-01-01

    Two mechanisms for chloroplast DNA replication have been revealed through the study of an unusual heteroplasmic strain of the green alga Chlamydomonas reinhardtii. Heteroplasmy is a state in which more than one genome type occurs in a mitochondrion or chloroplast. The Chlamydomonas strain spa19 bears two distinct chloroplast genomes, termed PS+ and PS−. PS+ genomes predominate and are stably maintained in vegetative cells, despite their lack of known replication origins. In sexual crosses with spa19 as the mating type plus parent, however, PS+ genomes are transmitted in only ∼25% of tetrads, whereas the PS− genomes are faithfully inherited in all progeny. In this research, we have explored the mechanism underlying this biased uniparental inheritance. We show that the relative reduction and dilution of PS+ vs. PS− genomes takes place during gametogenesis. Bromodeoxyuridine labeling, followed by immunoprecipitation and PCR, was used to compare replication activities of PS+ and PS− genomes. We found that the replication of PS+ genomes is specifically suppressed during gametogenesis and germination of zygospores, a phenomenon that also was observed when spa19 cells were treated with rifampicin, an inhibitor of the chloroplast RNA polymerase. Furthermore, when bromodeoxyuridine incorporation was compared at 11 sites within the chloroplast genome between vegetative cells, gametes, and rifampicin-treated cells by quantitative PCR, we found that incorporation was often reduced at the same sites in gametes that were also sensitive to rifampicin treatment. We conclude that a transcription-mediated form of DNA replication priming, which may be downregulated during gametogenesis, is indispensable for robust maintenance of PS+ genomes. These results highlight the potential for chloroplast genome copy number regulation through alternative replication strategies. PMID:20519744

  6. Differential replication of two chloroplast genome forms in heteroplasmic Chlamydomonas reinhardtii gametes contributes to alternative inheritance patterns.

    PubMed

    Nishimura, Yoshiki; Stern, David B

    2010-08-01

    Two mechanisms for chloroplast DNA replication have been revealed through the study of an unusual heteroplasmic strain of the green alga Chlamydomonas reinhardtii. Heteroplasmy is a state in which more than one genome type occurs in a mitochondrion or chloroplast. The Chlamydomonas strain spa19 bears two distinct chloroplast genomes, termed PS+ and PS-. PS+ genomes predominate and are stably maintained in vegetative cells, despite their lack of known replication origins. In sexual crosses with spa19 as the mating type plus parent, however, PS+ genomes are transmitted in only approximately 25% of tetrads, whereas the PS- genomes are faithfully inherited in all progeny. In this research, we have explored the mechanism underlying this biased uniparental inheritance. We show that the relative reduction and dilution of PS+ vs. PS- genomes takes place during gametogenesis. Bromodeoxyuridine labeling, followed by immunoprecipitation and PCR, was used to compare replication activities of PS+ and PS- genomes. We found that the replication of PS+ genomes is specifically suppressed during gametogenesis and germination of zygospores, a phenomenon that also was observed when spa19 cells were treated with rifampicin, an inhibitor of the chloroplast RNA polymerase. Furthermore, when bromodeoxyuridine incorporation was compared at 11 sites within the chloroplast genome between vegetative cells, gametes, and rifampicin-treated cells by quantitative PCR, we found that incorporation was often reduced at the same sites in gametes that were also sensitive to rifampicin treatment. We conclude that a transcription-mediated form of DNA replication priming, which may be downregulated during gametogenesis, is indispensable for robust maintenance of PS+ genomes. These results highlight the potential for chloroplast genome copy number regulation through alternative replication strategies.

  7. Alternative relay domains of Drosophila melanogaster myosin differentially affect ATPase activity, in vitro motility, myofibril structure and muscle function.

    PubMed

    Kronert, William A; Dambacher, Corey M; Knowles, Aileen F; Swank, Douglas M; Bernstein, Sanford I

    2008-06-06

    The relay domain of myosin is hypothesized to function as a communication pathway between the nucleotide-binding site, actin-binding site and the converter domain. In Drosophila melanogaster, a single myosin heavy chain gene encodes three alternative relay domains. Exon 9a encodes the indirect flight muscle isoform (IFI) relay domain, whereas exon 9b encodes one of the embryonic body wall isoform (EMB) relay domains. To gain a better understanding of the function of the relay domain and the differences imparted by the IFI and the EMB versions, we constructed two transgenic Drosophila lines expressing chimeric myosin heavy chains in indirect flight muscles lacking endogenous myosin. One expresses the IFI relay domain in the EMB backbone (EMB-9a), while the second expresses the EMB relay domain in the IFI backbone (IFI-9b). Our studies reveal that the EMB relay domain is functionally equivalent to the IFI relay domain when it is substituted into IFI. Essentially no differences in ATPase activity, actin-sliding velocity, flight ability at room temperature or muscle structure are observed in IFI-9b compared to native IFI. However, when the EMB relay domain is replaced with the IFI relay domain, we find a 50% reduction in actin-activated ATPase activity, a significant increase in actin affinity, abolition of actin sliding, defects in myofibril assembly and rapid degeneration of muscle structure compared to EMB. We hypothesize that altered relay domain conformational changes in EMB-9a impair intramolecular communication with the EMB-specific converter domain. This decreases transition rates involving strongly bound actomyosin states, leading to a reduced ATPase rate and loss of actin motility.

  8. Negative differential mobility for negative carriers as revealed by space charge measurements on crosslinked polyethylene insulated model cables

    SciTech Connect

    Teyssedre, G. Laurent, C.; Vu, T. T. N.

    2015-12-21

    Among features observed in polyethylene materials under relatively high field, space charge packets, consisting in a pulse of net charge that remains in the form of a pulse as it crosses the insulation, are repeatedly observed but without complete theory explaining their formation and propagation. Positive charge packets are more often reported, and the models based on negative differential mobility(NDM) for the transport of holes could account for some charge packets phenomenology. Conversely, NDM for electrons transport has never been reported so far. The present contribution reports space charge measurements by pulsed electroacoustic method on miniature cables that are model of HVDC cables. The measurements were realized at room temperature or with a temperature gradient of 10 °C through the insulation under DC fields on the order 30–60 kV/mm. Space charge results reveal systematic occurrence of a negative front of charges generated at the inner electrode that moves toward the outer electrode at the beginning of the polarization step. It is observed that the transit time of the front of negative charge increases, and therefore the mobility decreases, with the applied voltage. Further, the estimated mobility, in the range 10{sup −14}–10{sup −13} m{sup 2} V{sup −1} s{sup −1} for the present results, increases when the temperature increases for the same condition of applied voltage. The features substantiate the hypothesis of negative differential mobility used for modelling space charge packets.

  9. Extracellular matrix of adipogenically differentiated mesenchymal stem cells reveals a network of collagen filaments, mostly interwoven by hexagonal structural units.

    PubMed

    Ullah, Mujib; Sittinger, Michael; Ringe, Jochen

    2013-01-01

    Extracellular matrix (ECM) is the non-cellular component of tissues, which not only provides biological shelter but also takes part in the cellular decisions for diverse functions. Every tissue has an ECM with unique composition and topology that governs the process of determination, differentiation, proliferation, migration and regeneration of cells. Little is known about the structural organization of matrix especially of MSC-derived adipogenic ECM. Here, we particularly focus on the composition and architecture of the fat ECM to understand the cellular behavior on functional bases. Thus, mesenchymal stem cells (MSC) were adipogenically differentiated, then, were transferred to adipogenic propagation medium, whereas they started the release of lipid droplets leaving bare network of ECM. Microarray analysis was performed, to indentify the molecular machinery of matrix. Adipogenesis was verified by Oil Red O staining of lipid droplets and by qPCR of adipogenic marker genes PPARG and FABP4. Antibody staining demonstrated the presence of collagen type I, II and IV filaments, while alkaline phosphatase activity verified the ossified nature of these filaments. In the adipogenic matrix, the hexagonal structures were abundant followed by octagonal structures, whereas they interwoven in a crisscross manner. Regarding molecular machinery of adipogenic ECM, the bioinformatics analysis revealed the upregulated expression of COL4A1, ITGA7, ITGA7, SDC2, ICAM3, ADAMTS9, TIMP4, GPC1, GPC4 and downregulated expression of COL14A1, ADAMTS5, TIMP2, TIMP3, BGN, LAMA3, ITGA2, ITGA4, ITGB1, ITGB8, CLDN11. Moreover, genes associated with integrins, glycoproteins, laminins, fibronectins, cadherins, selectins and linked signaling pathways were found. Knowledge of the interactive-language between cells and matrix could be beneficial for the artificial designing of biomaterials and bioscaffolds.

  10. Latitudinal gradients in tree ring stable carbon and oxygen isotopes reveal differential climate influences of the North American Monsoon System

    NASA Astrophysics Data System (ADS)

    Szejner, Paul; Wright, William E.; Babst, Flurin; Belmecheri, Soumaya; Trouet, Valerie; Leavitt, Steven W.; Ehleringer, James R.; Monson, Russell K.

    2016-07-01

    The arrival of the North American Monsoon System (NAMS) terminates a presummer hyperarid period in the southwestern United States (U.S.), providing summer moisture that is favorable for forest growth. Montane forests in this region rely on winter snowpack to drive much of their growth; the extent to which they use NAMS moisture is uncertain. We addressed this by studying stable carbon and oxygen isotopes in earlywood and latewood from 11 sites along a latitudinal gradient extending from Arizona and New Mexico to Utah. This study provides the first regional perspective on the relative roles of winter versus summer precipitation as an ecophysiological resource. Here we present evidence that Ponderosa pine uses NAMS moisture differentially across this gradient. 13C/12C ratios suggest that photosynthetic water use efficiency during latewood formation is more sensitive to summer precipitation at the northern than at the southern sites. This is likely due to the fact that NAMS moisture provides sufficiently favorable conditions for tree photosynthesis and growth during most years in the southern sites, whereas the northern sites experience larger summer moisture variability, which in some years is limiting growth. Cellulose δ18O and δ13C values revealed that photoassimilates in the southern sites were produced under higher vapor pressure deficit conditions during spring compared to summer, demonstrating a previously underappreciated effect of seasonal differences in atmospheric humidity on tree ring isotope ratios. Our findings suggest that future changes in NAMS will potentially alter productivity and photosynthetic water use dynamics differentially along latitudinal gradients in southwestern U.S. montane forests.

  11. "Contrasting patterns of selection at Pinus pinaster Ait. Drought stress candidate genes as revealed by genetic differentiation analyses".

    PubMed

    Eveno, Emmanuelle; Collada, Carmen; Guevara, M Angeles; Léger, Valérie; Soto, Alvaro; Díaz, Luis; Léger, Patrick; González-Martínez, Santiago C; Cervera, M Teresa; Plomion, Christophe; Garnier-Géré, Pauline H

    2008-02-01

    The importance of natural selection for shaping adaptive trait differentiation among natural populations of allogamous tree species has long been recognized. Determining the molecular basis of local adaptation remains largely unresolved, and the respective roles of selection and demography in shaping population structure are actively debated. Using a multilocus scan that aims to detect outliers from simulated neutral expectations, we analyzed patterns of nucleotide diversity and genetic differentiation at 11 polymorphic candidate genes for drought stress tolerance in phenotypically contrasted Pinus pinaster Ait. populations across its geographical range. We compared 3 coalescent-based methods: 2 frequentist-like, including 1 approach specifically developed for biallelic single nucleotide polymorphisms (SNPs) here and 1 Bayesian. Five genes showed outlier patterns that were robust across methods at the haplotype level for 2 of them. Two genes presented higher F(ST) values than expected (PR-AGP4 and erd3), suggesting that they could have been affected by the action of diversifying selection among populations. In contrast, 3 genes presented lower F(ST) values than expected (dhn-1, dhn2, and lp3-1), which could represent signatures of homogenizing selection among populations. A smaller proportion of outliers were detected at the SNP level suggesting the potential functional significance of particular combinations of sites in drought-response candidate genes. The Bayesian method appeared robust to low sample sizes, flexible to assumptions regarding migration rates, and powerful for detecting selection at the haplotype level, but the frequentist-like method adapted to SNPs was more efficient for the identification of outlier SNPs showing low differentiation. Population-specific effects estimated in the Bayesian method also revealed populations with lower immigration rates, which could have led to favorable situations for local adaptation. Outlier patterns are discussed

  12. Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea

    PubMed Central

    2013-01-01

    Background Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. Results We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. Conclusion The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that

  13. Flux balance analysis reveals acetate metabolism modulates cyclic electron flow and alternative glycolytic pathways in Chlamydomonas reinhardtii.

    PubMed

    Chapman, Stephen P; Paget, Caroline M; Johnson, Giles N; Schwartz, Jean-Marc

    2015-01-01

    Cells of the green alga Chlamydomonas reinhardtii cultured in the presence of acetate perform mixotrophic growth, involving both photosynthesis and organic carbon assimilation. Under such conditions, cells exhibit a reduced capacity for photosynthesis but a higher growth rate, compared to phototrophic cultures. Better understanding of the down regulation of photosynthesis would enable more efficient conversion of carbon into valuable products like biofuels. In this study, Flux Balance Analysis (FBA) and Flux Variability Analysis (FVA) have been used with a genome scale model of C. reinhardtii to examine changes in intracellular flux distribution in order to explain their changing physiology. Additionally, a reaction essentiality analysis was performed to identify which reaction subsets are essential for a given growth condition. Our results suggest that exogenous acetate feeds into a modified tricarboxylic acid (TCA) cycle, which bypasses the CO2 evolution steps, explaining increases in biomass, consistent with experimental data. In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions. Importantly, acetate addition leads to an increased flux through cyclic electron flow (CEF), but results in a repression of CO2 fixation via Rubisco, explaining the down regulation of photosynthesis. However, although CEF enhances growth on acetate, it is not essential-impairment of CEF results in alternative metabolic pathways being increased. We have demonstrated how the reactions of photosynthesis interconnect with carbon metabolism on a global scale, and how systems approaches play a viable tool in understanding complex relationships at the scale of the organism.

  14. Genetic evidence reveals density-dependent mediated success of alternative mating behaviours in the European bitterling (Rhodeus sericeus).

    PubMed

    Reichard, M; Smith, C; Jordan, W C

    2004-06-01

    The reproductive success of alternative mating behaviours may vary within and among populations in relation to environmental factors and demographic parameters. We used behavioural and genetic data to investigate how male density affects reproductive success of territoriality and sneaking in the European bitterling (Rhodeus sericeus, Cyprinidae), a freshwater fish that spawns on the gills of living freshwater mussels. Keeping the number of spawning sites constant, we manipulated male densities in laboratory and mesocosm experiments. We showed that sneaked fertilizations were common in R. sericeus, and that they increased significantly with male density. Territorial mating was almost 17 times more successful than sneaking at the lowest male density treatment, and still 2-3 times more successful at intermediate densities. However, both behaviours conferred the same fitness pay-off at the highest male density. While the success of territorial males declined with male density, the success of individual sneaking males remained constant across densities. Notably, the capacity of territorial males to outcompete sneakers by preoviposition sperm loading was the best predictor of male reproductive success, rather than aggression, body size or postoviposition ejaculation.

  15. Flux balance analysis reveals acetate metabolism modulates cyclic electron flow and alternative glycolytic pathways in Chlamydomonas reinhardtii

    PubMed Central

    Chapman, Stephen P.; Paget, Caroline M.; Johnson, Giles N.; Schwartz, Jean-Marc

    2015-01-01

    Cells of the green alga Chlamydomonas reinhardtii cultured in the presence of acetate perform mixotrophic growth, involving both photosynthesis and organic carbon assimilation. Under such conditions, cells exhibit a reduced capacity for photosynthesis but a higher growth rate, compared to phototrophic cultures. Better understanding of the down regulation of photosynthesis would enable more efficient conversion of carbon into valuable products like biofuels. In this study, Flux Balance Analysis (FBA) and Flux Variability Analysis (FVA) have been used with a genome scale model of C. reinhardtii to examine changes in intracellular flux distribution in order to explain their changing physiology. Additionally, a reaction essentiality analysis was performed to identify which reaction subsets are essential for a given growth condition. Our results suggest that exogenous acetate feeds into a modified tricarboxylic acid (TCA) cycle, which bypasses the CO2 evolution steps, explaining increases in biomass, consistent with experimental data. In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions. Importantly, acetate addition leads to an increased flux through cyclic electron flow (CEF), but results in a repression of CO2 fixation via Rubisco, explaining the down regulation of photosynthesis. However, although CEF enhances growth on acetate, it is not essential—impairment of CEF results in alternative metabolic pathways being increased. We have demonstrated how the reactions of photosynthesis interconnect with carbon metabolism on a global scale, and how systems approaches play a viable tool in understanding complex relationships at the scale of the organism. PMID:26175742

  16. Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation

    PubMed Central

    Schmidt, Ute; Robert, Marie-Cécile; Yoshida, Minoru; Villemin, Jean-Philippe; Auboeuf, Didier; Aitken, Stuart

    2011-01-01

    Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2–GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules. PMID:21624952

  17. Single-nucleotide resolution mapping of the Gossypium raimondii transcriptome reveals a new mechanism for alternative splicing of introns.

    PubMed

    Li, Qin; Xiao, Guanghui; Zhu, Yu-Xian

    2014-05-01

    Alternative splicing (AS) is a vital genetic mechanism that enhances the diversity of eukaryotic transcriptomes. Here, we generated 8.3 Gb high-quality RNA-sequencing data from cotton (Gossypium raimondii) and performed a systematic, comparative analysis of AS events. We mapped 85% of the RNA-sequencing data onto the reference genome and identified 154368 splice junctions with 16437 as events in 10197 genes. Intron retention constituted the majority (40%) of all AS events in G. raimondii. Comparison across 11 eukaryote species showed that intron retention is the most common AS type in higher plants. Although transposable elements (TEs) were found in only 2.9% of all G. raimondii introns, they are present in 43% of the retained introns, suggesting that TE-insertion may be an important mechanism for intron retention during AS. The majority of the TE insertions are concentrated 0-40 nt upstream of the 3'-splice site, substantially altering the distribution of branch points from preferred positions and reducing the efficiency of intron splicing by decreasing RNA secondary structure flexibility. Our data suggest that TE-insertion-induced changes in branch point-site distribution are important for intron retention-type AS. Our findings may help explain the vast differences in intron-retention frequencies between vertebrates and higher plants.

  18. Genome-wide regulatory analysis reveals T-bet controls Th17 lineage differentiation through direct suppression of IRF41

    PubMed Central

    Gökmen, M. Refik; Dong, Rong; Kanhere, Aditi; Powell, Nick; Perucha, Esperanza; Jackson, Ian; Howard, Jane K.; Hernandez-Fuentes, Maria; Jenner, Richard G.; Lord, Graham M.

    2013-01-01

    The complex relationship between Th1 and Th17 cells is incompletely understood. The transcription factor T-bet is best known as the master regulator of Th1 lineage commitment. However, attention is now focused on the repression of alternate T cell subsets mediated by T-bet, particularly the Th17 lineage. It has recently been suggested that pathogenic Th17 cells express T-bet and are dependent on IL-23. However, T-bet has previously been shown to be a negative regulator of Th17 cells. We have taken an unbiased approach to determine the functional impact of T-bet on Th17 lineage commitment. Genome-wide analysis of functional T-bet binding sites provides an improved understanding of the transcriptional regulation mediated by T-bet, and suggests novel mechanisms by which T-bet regulates T helper cell differentiation. Specifically, we show that T-bet negatively regulates Th17 lineage commitment via direct repression of the transcription factor interferon regulatory factor-4 (IRF4). An in vivo analysis of the pathogenicity of T-bet deficient T cells demonstrated that mucosal Th17 responses were augmented in the absence of T-bet, and we have demonstrated that the role of T-bet in enforcing Th1 responses and suppressing Th17 responses are separable. The interaction of the two key transcription factors T-bet and IRF4 during the determination of T cell fate choice significantly advances our understanding of the mechanisms underlying the development of pathogenic T cells. PMID:24249732

  19. A classification scheme for alternative oxidases reveals the taxonomic distribution and evolutionary history of the enzyme in angiosperms.

    PubMed

    Costa, José Hélio; McDonald, Allison E; Arnholdt-Schmitt, Birgit; Fernandes de Melo, Dirce

    2014-11-01

    A classification scheme based on protein phylogenies and sequence harmony method was used to clarify the taxonomic distribution and evolutionary history of the alternative oxidase (AOX) in angiosperms. A large data set analyses showed that AOX1 and AOX2 subfamilies were distributed into 4 phylogenetic clades: AOX1a-c/1e, AOX1d, AOX2a-c and AOX2d. High diversity in AOX family compositions was found. While the AOX2 subfamily was not detected in monocots, the AOX1 subfamily has expanded (AOX1a-e) in the large majority of these plants. In addition, Poales AOX1b and 1d were orthologous to eudicots AOX1d and then renamed as AOX1d1 and 1d2. AOX1 or AOX2 losses were detected in some eudicot plants. Several AOX2 duplications (AOX2a-c) were identified in eudicot species, mainly in the asterids. The AOX2b originally identified in eudicots in the Fabales order (soybean, cowpea) was divergent from AOX2a-c showing some specific amino acids with AOX1d and then it was renamed as AOX2d. AOX1d and AOX2d seem to be stress-responsive, facultative and mutually exclusive among species suggesting a complementary role with an AOX1(a) in stress conditions. Based on the data collected, we present a model for the evolutionary history of AOX in angiosperms and highlight specific areas where further research would be most beneficial.

  20. MtDNA meta-analysis reveals both phenotype specificity and allele heterogeneity: a model for differential association

    PubMed Central

    Marom, Shani; Friger, Michael; Mishmar, Dan

    2017-01-01

    Human mtDNA genetic variants have traditionally been considered markers for ancient population migrations. However, during the past three decades, these variants have been associated with altered susceptibility to various phenotypes, thus supporting their importance for human health. Nevertheless, mtDNA disease association has frequently been supported only in certain populations, due either to population stratification or differential epistatic compensations among populations. To partially overcome these obstacles, we performed meta-analysis of the multiple mtDNA association studies conducted until 2016, encompassing 53,975 patients and 63,323 controls. Our findings support the association of mtDNA haplogroups and recurrent variants with specific phenotypes such as Parkinson’s disease, type 2 diabetes, longevity, and breast cancer. Strikingly, our assessment of mtDNA variants’ involvement with multiple phenotypes revealed significant impact for Caucasian haplogroups H, J, and K. Therefore, ancient mtDNA variants could be divided into those that affect specific phenotypes, versus others with a general impact on phenotype combinations. We suggest that the mtDNA could serve as a model for phenotype specificity versus allele heterogeneity. PMID:28230165

  1. Differential Proteomic Analysis by iTRAQ Reveals the Mechanism of Pyropia haitanensis Responding to High Temperature Stress

    PubMed Central

    Shi, Jianzhi; Chen, Yuting; Xu, Yan; Ji, Dehua; Chen, Changsheng; Xie, Chaotian

    2017-01-01

    Global warming increases sea temperature and leads to high temperature stress, which affects the yield and quality of Pyropia haitanensis. To understand the molecular mechanisms underlying high temperature stress in a high temperature tolerance strain Z-61, the iTRAQ technique was employed to reveal the global proteomic response of Z-61 under different durations of high temperature stress. We identified 151 differentially expressed proteins and classified them into 11 functional categories. The 4 major categories of these are protein synthesis and degradation, photosynthesis, defense response, and energy and carbohydrate metabolism. These findings indicated that photosynthesis, protein synthesis, and secondary metabolism are inhibited by heat to limit damage to a repairable level. As time progresses, misfolded proteins and ROS accumulate and lead to the up-regulation of molecular chaperones, proteases, and antioxidant systems. Furthermore, to cope with cells injured by heat, PCD works to remove them. Additionally, sulfur assimilation and cytoskeletons play essential roles in maintaining cellular and redox homeostasis. These processes are based on signal transduction in the phosphoinositide pathway and multiple ways to supply energy. Conclusively, Z-61 establishes a new steady-state balance of metabolic processes and survives under higher temperature stress. PMID:28303955

  2. Differential Proteomic Analysis by iTRAQ Reveals the Mechanism of Pyropia haitanensis Responding to High Temperature Stress.

    PubMed

    Shi, Jianzhi; Chen, Yuting; Xu, Yan; Ji, Dehua; Chen, Changsheng; Xie, Chaotian

    2017-03-17

    Global warming increases sea temperature and leads to high temperature stress, which affects the yield and quality of Pyropia haitanensis. To understand the molecular mechanisms underlying high temperature stress in a high temperature tolerance strain Z-61, the iTRAQ technique was employed to reveal the global proteomic response of Z-61 under different durations of high temperature stress. We identified 151 differentially expressed proteins and classified them into 11 functional categories. The 4 major categories of these are protein synthesis and degradation, photosynthesis, defense response, and energy and carbohydrate metabolism. These findings indicated that photosynthesis, protein synthesis, and secondary metabolism are inhibited by heat to limit damage to a repairable level. As time progresses, misfolded proteins and ROS accumulate and lead to the up-regulation of molecular chaperones, proteases, and antioxidant systems. Furthermore, to cope with cells injured by heat, PCD works to remove them. Additionally, sulfur assimilation and cytoskeletons play essential roles in maintaining cellular and redox homeostasis. These processes are based on signal transduction in the phosphoinositide pathway and multiple ways to supply energy. Conclusively, Z-61 establishes a new steady-state balance of metabolic processes and survives under higher temperature stress.

  3. Mass spectrometric phosphoproteome analysis of HIV-infected brain reveals novel phosphorylation sites and differential phosphorylation patterns

    PubMed Central

    Uzasci, Lerna; Auh, Sungyoung; Cotter, Robert J.; Nath, Avindra

    2016-01-01

    Purpose To map the phosphoproteome and identify changes in the phosphorylation patterns in the HIV-infected and uninfected brain using high-resolution mass spectrometry. Experimental Design Parietal cortex from brain of individuals with and without HIV infection were lysed and trypsinized. The peptides were labeled with iTRAQ reagents, combined, phospho-enriched by titanium dioxide chromatography, and analyzed by LC-MS/MS with high-resolution. Results Our phosphoproteomic workflow resulted in the identification of 112 phosphorylated proteins and 17 novel phosphorylation sites in all the samples that were analyzed. The phosphopeptide sequences were searched for kinase substrate motifs which revealed potential kinases involved in important signaling pathways. The site-specific phosphopeptide quantification showed that peptides from neurofilament medium polypeptide, myelin basic protein, and 2′–3′-cyclic nucleotide-3′ phosphodiesterase have relatively higher phosphorylation levels during HIV infection. Clinical Relevance This study has enriched the global phosphoproteome knowledge of the human brain by detecting novel phosphorylation sites on neuronal proteins and identifying differentially phosphorylated brain proteins during HIV infection. Kinases that lead to unusual phosphorylations could be therapeutic targets for the treatment of HIV-associated neurocognitive disorders (HAND). PMID:26033855

  4. Proteome profiling of the growth phases of Leishmania pifanoi promastigotes in axenic culture reveals differential abundance of immunostimulatory proteins.

    PubMed

    Alcolea, Pedro J; Alonso, Ana; García-Tabares, Francisco; Mena, María del Carmen; Ciordia, Sergio; Larraga, Vicente

    2016-06-01

    Leishmaniasis is a term that encompasses a compendium of neglected tropical diseases caused by dimorphic and digenetic protozoan parasites from the genus Leishmania (Kinetoplastida: Trypanosomatidae). The clinical manifestations of neotropical cutaneous leishmaniasis (NCL) caused by Leishmania pifanoi and other species of the "Leishmania mexicana complex" mainly correspond to anergic diffuse cutaneous leishmaniasis (ADCL), which is the origin of considerable morbidity. Despite the outstanding advances in the characterization of the trypanosomatid genomes and proteomes, the biology of this species has been scarcely explored. However, the close relation of L. pifanoi to the sequenced species L. mexicana and others included in the "L. mexicana complex" allowed us to perform a two-dimension electrophoresis (2DE) approach to the promastigote proteome at the differential expression level. Protein identifications were performed by matrix-assisted laser desorption-ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). This insight has revealed similarities and differences between L. pifanoi and other species responsible for cutaneous and visceral leishmaniasis. Interestingly, certain proteins that were previously described as immunostimulatory (elongation factor 1β, trypanothione peroxidase, heat shock protein 70, enolase, GDP-forming succinyl-CoA and aldehyde dehydrogenase) are more abundant in the final growth stages of promastigotes (late-logarithmic and/or stationary phase) in the case of L. pifanoi.

  5. High Differentiation among Eight Villages in a Secluded Area of Sardinia Revealed by Genome-Wide High Density SNPs Analysis

    PubMed Central

    Pirastu, Nicola; Persico, Ivana; Sassu, Alessandro; Picciau, Andrea; Prodi, Dionigio; Fraumene, Cristina; Mocci, Evelina; Manias, Maria Teresa; Atzeni, Rossano; Cosso, Massimiliano; Pirastu, Mario

    2009-01-01

    To better design association studies for complex traits in isolated populations it's important to understand how history and isolation moulded the genetic features of different communities. Population isolates should not “a priori” be considered homogeneous, even if the communities are not distant and part of a small region. We studied a particular area of Sardinia called Ogliastra, characterized by the presence of several distinct villages that display different history, immigration events and population size. Cultural and geographic isolation characterized the history of these communities. We determined LD parameters in 8 villages and defined population structure through high density SNPs (about 360 K) on 360 unrelated people (45 selected samples from each village). These isolates showed differences in LD values and LD map length. Five of these villages show high LD values probably due to their reduced population size and extreme isolation. High genetic differentiation among villages was detected. Moreover population structure analysis revealed a high correlation between genetic and geographic distances. Our study indicates that history, geography and biodemography have influenced the genetic features of Ogliastra communities producing differences in LD and population structure. All these data demonstrate that we can consider each village an isolate with specific characteristics. We suggest that, in order to optimize the study design of complex traits, a thorough characterization of genetic features is useful to identify the presence of sub-populations and stratification within genetic isolates. PMID:19247500

  6. Transcriptome analysis reveals regulatory networks underlying differential susceptibility to Botrytis cinerea in response to nitrogen availability in Solanum lycopersicum

    PubMed Central

    Vega, Andrea; Canessa, Paulo; Hoppe, Gustavo; Retamal, Ignacio; Moyano, Tomas C.; Canales, Javier; Gutiérrez, Rodrigo A.; Rubilar, Joselyn

    2015-01-01

    Nitrogen (N) is one of the main limiting nutrients for plant growth and crop yield. It is well documented that changes in nitrate availability, the main N source found in agricultural soils, influences a myriad of developmental programs and processes including the plant defense response. Indeed, many agronomical reports indicate that the plant N nutritional status influences their ability to respond effectively when challenged by different pathogens. However, the molecular mechanisms involved in N-modulation of plant susceptibility to pathogens are poorly characterized. In this work, we show that Solanum lycopersicum defense response to the necrotrophic fungus Botrytis cinerea is affected by plant N availability, with higher susceptibility in nitrate-limiting conditions. Global gene expression responses of tomato against B. cinerea under contrasting nitrate conditions reveals that plant primary metabolism is affected by the fungal infection regardless of N regimes. This result suggests that differential susceptibility to pathogen attack under contrasting N conditions is not only explained by a metabolic alteration. We used a systems biology approach to identify the transcriptional regulatory network implicated in plant response to the fungus infection under contrasting nitrate conditions. Interestingly, hub genes in this network are known key transcription factors involved in ethylene and jasmonic acid signaling. This result positions these hormones as key integrators of nitrate and defense against B. cinerea in tomato plants. Our results provide insights into potential crosstalk mechanisms between necrotrophic defense response and N status in plants. PMID:26583019

  7. Host and non-host roots in rice: cellular and molecular approaches reveal differential responses to arbuscular mycorrhizal fungi

    PubMed Central

    Fiorilli, Valentina; Vallino, Marta; Biselli, Chiara; Faccio, Antonella; Bagnaresi, Paolo; Bonfante, Paola

    2015-01-01

    Oryza sativa, a model plant for Arbuscular Mycorrhizal (AM) symbiosis, has both host and non-host roots. Large lateral (LLR) and fine lateral (FLR) roots display opposite responses: LLR support AM colonization, but FLR do not. Our research aimed to study the molecular, morphological and physiological aspects related to the non-host behavior of FLR. RNA-seq analysis revealed that LLR and FLR displayed divergent expression profiles, including changes in many metabolic pathways. Compared with LLR, FLR showed down-regulation of genes instrumental for AM establishment and gibberellin signaling, and a higher expression of nutrient transporters. Consistent with the transcriptomic data, FLR had higher phosphorus content. Light and electron microscopy demonstrated that, surprisingly, in the Selenio cultivar, FLR have a two-layered cortex, which is theoretically compatible with AM colonization. According to RNA-seq, a gibberellin inhibitor treatment increased anticlinal divisions leading to a higher number of cortex cells in FLR. We propose that some of the differentially regulated genes that lead to the anatomical and physiological properties of the two root types also function as genetic factors regulating fungal colonization. The rice root apparatus offers a unique tool to study AM symbiosis, allowing direct comparisons of host and non-host roots in the same individual plant. PMID:26322072

  8. Morphology informed by phylogeny reveals unexpected patterns of species differentiation in the aquatic moss Rhynchostegium riparioides s.l.

    PubMed

    Hutsemékers, Virginie; Vieira, Cristiana C; Ros, Rosa María; Huttunen, Sanna; Vanderpoorten, Alain

    2012-02-01

    Bryophyte floras typically exhibit extremely low levels of endemism. The interpretation, that this might reflect taxonomic shortcomings, is tested here for the Macaronesian flora, using the moss species complex of Rhynchostegium riparioides as a model. The deep polyphyly of R. riparioides across its distribution range reveals active differentiation that better corresponds to geographic than morphological differences. Morphometric analyses are, in fact, blurred by a size gradient that accounts for 80% of the variation observed among gametophytic traits. The lack of endemic diversification observed in R. riparioides in Macaronesia weakens the idea that the low rates of endemism observed in the Macaronesian bryophyte flora might solely be explained by taxonomic shortcomings. To the reverse, the striking polyphyly of North American and European lineages of R. riparioides suggests that the similarity between the floras of these continents has been over-emphasized. Discriminant analyses point to the existence of morphological discontinuities among the lineages resolved by the molecular phylogeny. The global rate of error associated to species identification based on morphology (0.23) indicates, however, that intergradation of shape and size characters among species in the group challenges their identification.

  9. Pronounced genetic differentiation and recent secondary contact in the mangrove tree Lumnitzera racemosa revealed by population genomic analyses

    PubMed Central

    Li, Jianfang; Yang, Yuchen; Chen, Qipian; Fang, Lu; He, Ziwen; Guo, Wuxia; Qiao, Sitan; Wang, Zhengzhen; Guo, Miaomiao; Zhong, Cairong; Zhou, Renchao; Shi, Suhua

    2016-01-01

    Systematically investigating the impacts of Pleistocene sea-level fluctuations on mangrove plants may provide a better understanding of their demographic history and useful information for their conservation. Therefore, we conducted population genomic analyses of 88 nuclear genes to explore the population dynamics of a mangrove tree Lumnitzera racemosa across the Indo-West Pacific region. Our results revealed pronounced genetic differentiation in this species between the populations from the Indian Ocean and the Pacific Ocean, which may be attributable to the long-term isolation between the western and eastern coasts of the Malay Peninsula during sea-level drops in the Pleistocene glacial periods. The mixing of haplotypes from the two highly divergent groups was identified in a Cambodian population at almost all 88 nuclear genes, suggesting genetic admixture of the two lineages at the boundary region. Similar genetic admixture was also found in other populations from Southeast Asia based on the Bayesian clustering analysis of six nuclear genes, which suggests extensive and recent secondary contact of the two divergent lineages in Southeast Asia. Computer simulations indicated substantial migration from the Indian Ocean towards the South China Sea, which likely results in the genetic admixture in Southeast Asia. PMID:27380895

  10. Differential nuclease sensitivity profiling of chromatin reveals biochemical footprints coupled to gene expression and functional DNA elements in maize.

    PubMed

    Vera, Daniel L; Madzima, Thelma F; Labonne, Jonathan D; Alam, Mohammad P; Hoffman, Gregg G; Girimurugan, S B; Zhang, Jinfeng; McGinnis, Karen M; Dennis, Jonathan H; Bass, Hank W

    2014-10-01

    The eukaryotic genome is organized into nucleosomes, the fundamental units of chromatin. The positions of nucleosomes on DNA regulate protein-DNA interactions and in turn influence DNA-templated events. Despite the increasing number of genome-wide maps of nucleosome position, how global changes in gene expression relate to changes in nucleosome position is poorly understood. We show that in nucleosome occupancy mapping experiments in maize (Zea mays), particular genomic regions are highly susceptible to variation introduced by differences in the extent to which chromatin is digested with micrococcal nuclease (MNase). We exploited this digestion-linked variation to identify protein footprints that are hypersensitive to MNase digestion, an approach we term differential nuclease sensitivity profiling (DNS-chip). Hypersensitive footprints were enriched at the 5' and 3' ends of genes, associated with gene expression levels, and significantly overlapped with conserved noncoding sequences and the binding sites of the transcription factor KNOTTED1. We also found that the tissue-specific regulation of gene expression was linked to tissue-specific hypersensitive footprints. These results reveal biochemical features of nucleosome organization that correlate with gene expression levels and colocalize with functional DNA elements. This approach to chromatin profiling should be broadly applicable to other species and should shed light on the relationships among chromatin organization, protein-DNA interactions, and genome regulation.

  11. Changes in localization of human discs large (hDlg) during keratinocyte differentiation is associated with expression of alternatively spliced hDlg variants

    SciTech Connect

    Roberts, S. . E-mail: s.roberts@bham.ac.uk; Calautti, E.; Vanderweil, S.; Nguyen, H.O.; Foley, A.; Baden, H.P.; Viel, A.

    2007-07-15

    Alternative spliced variants of the human discs large (hDlg) tumour suppressor are characterized by combinations of insertions. Here, using insertions I2- and I3-specific antibodies, we show that I2 and I3 variants have distinct distributions in epidermal and cervical epithelia. In skin and cervix, I3 variants are found in the cytoplasm. Cytoplasmic localization of I3 variants decreases as cervical keratinocytes differentiate, concomitant with relocalization to the cell periphery. I2 variants are found at the cell periphery of differentiated epidermal and cervical keratinocytes. Nuclear localization of I2 variants was evident in both tissues, with concentration of nuclear I2 variants in basal and parabasal cervical keratinocytes. A prominent nuclear localization of hDlg in cells of hyperproliferative layers of psoriatic lesions, but not in mature differentiated keratinocytes, together with I2 redistribution in differentiating keratinocytes, suggests that nuclear hDlg functions may be pertinent to growth of undifferentiated cells. Supporting our findings in squamous tissues, a decrease of nuclear hDlg and an increase of membrane-bound and cytoplasmic hDlg upon calcium-induced keratinocyte differentiation were not concomitant processes. Furthermore, we confirm that the exit of I2 variants from the nucleus is linked to stimulation of epithelial differentiation. The dynamic redistribution of hDlg also correlated with a marked increase in the expression of I3 variants while the level of I2 variants showed only a moderate decrease. Because changes in the intracellular distribution of hDlg splice variants, and in their expression levels, correlate with changes in differentiation state we hypothesize that the different hDlg isoforms play distinct roles at various stages of epithelial differentiation.

  12. Alternative Isoform Analysis of Ttc8 Expression in the Rat Pineal Gland Using a Multi-Platform Sequencing Approach Reveals Neural Regulation.

    PubMed

    Hartley, Stephen W; Mullikin, James C; Klein, David C; Park, Morgan; Coon, Steven L

    Alternative isoform regulation (AIR) vastly increases transcriptome diversity and plays an important role in numerous biological processes and pathologies. However, the detection and analysis of isoform-level differential regulation is difficult, particularly in the face of complex and incompletely-annotated transcriptomes. Here we have used Illumina short-read/high-throughput RNA-Seq to identify 55 genes that exhibit neurally-regulated AIR in the pineal gland, and then used two other complementary experimental platforms to further study and characterize the Ttc8 gene, which is involved in Bardet-Biedl syndrome and non-syndromic retinitis pigmentosa. Use of the JunctionSeq analysis tool led to the detection of several novel exons and splice junctions in this gene, including two novel alternative transcription start sites which were found to display disproportionately strong neurally-regulated differential expression in several independent experiments. These high-throughput sequencing results were validated and augmented via targeted qPCR and long-read Pacific Biosciences SMRT sequencing. We confirmed the existence of numerous novel splice junctions and the selective upregulation of the two novel start sites. In addition, we identified more than 20 novel isoforms of the Ttc8 gene that are co-expressed in this tissue. By using information from multiple independent platforms we not only greatly reduce the risk of errors, biases, and artifacts influencing our results, we also are able to characterize the regulation and splicing of the Ttc8 gene more deeply and more precisely than would be possible via any single platform. The hybrid method outlined here represents a powerful strategy in the study of the transcriptome.

  13. Alternative Isoform Analysis of Ttc8 Expression in the Rat Pineal Gland Using a Multi-Platform Sequencing Approach Reveals Neural Regulation

    PubMed Central

    Mullikin, James C.; Klein, David C.; Park, Morgan; Coon, Steven L.

    2016-01-01

    Alternative isoform regulation (AIR) vastly increases transcriptome diversity and plays an important role in numerous biological processes and pathologies. However, the detection and analysis of isoform-level differential regulation is difficult, particularly in the face of complex and incompletely-annotated transcriptomes. Here we have used Illumina short-read/high-throughput RNA-Seq to identify 55 genes that exhibit neurally-regulated AIR in the pineal gland, and then used two other complementary experimental platforms to further study and characterize the Ttc8 gene, which is involved in Bardet-Biedl syndrome and non-syndromic retinitis pigmentosa. Use of the JunctionSeq analysis tool led to the detection of several novel exons and splice junctions in this gene, including two novel alternative transcription start sites which were found to display disproportionately strong neurally-regulated differential expression in several independent experiments. These high-throughput sequencing results were validated and augmented via targeted qPCR and long-read Pacific Biosciences SMRT sequencing. We confirmed the existence of numerous novel splice junctions and the selective upregulation of the two novel start sites. In addition, we identified more than 20 novel isoforms of the Ttc8 gene that are co-expressed in this tissue. By using information from multiple independent platforms we not only greatly reduce the risk of errors, biases, and artifacts influencing our results, we also are able to characterize the regulation and splicing of the Ttc8 gene more deeply and more precisely than would be possible via any single platform. The hybrid method outlined here represents a powerful strategy in the study of the transcriptome. PMID:27684375

  14. Alternative Splicing Studies of the Reactive Oxygen Species Gene Network in Populus Reveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase1[C][W

    PubMed Central

    Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

    2009-01-01

    Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays. PMID:19176719

  15. Masked priming of conceptual features reveals differential brain activation during unconscious access to conceptual action and sound information.

    PubMed

    Trumpp, Natalie M; Traub, Felix; Kiefer, Markus

    2013-01-01

    Previous neuroimaging studies suggested an involvement of sensory-motor brain systems during conceptual processing in support of grounded cognition theories of conceptual memory. However, in these studies with visible stimuli, contributions of strategic imagery or semantic elaboration processes to observed sensory-motor activity cannot be entirely excluded. In the present study, we therefore investigated the electrophysiological correlates of unconscious feature-specific priming of action- and sound-related concepts within a novel feature-priming paradigm to specifically probe automatic processing of conceptual features without the contribution of possibly confounding factors such as orthographic similarity or response congruency. Participants were presented with a masked subliminal prime word and a subsequent visible target word. In the feature-priming conditions primes as well as targets belonged to the same conceptual feature dimension (action or sound, e.g., typewriter or radio) whereas in the two non-priming conditions, either the primes or the targets consisted of matched control words with low feature relevance (e.g., butterfly or candle). Event-related potential analyses revealed unconscious feature-specific priming effects at fronto-central electrodes within 100 to 180 ms after target stimulus onset that differed with regard to topography and underlying neural generators. In congruency with previous findings under visible stimulation conditions, these differential subliminal ERP feature-priming effects demonstrate an unconscious automatic access to action versus sound features of concepts. The present results therefore support grounded cognition theory suggesting that activity in sensory and motor areas during conceptual processing can also occur unconsciously and is not mandatorily accompanied by a vivid conscious experience of the conceptual content such as in imagery.

  16. Multilocus sequence analysis of Thermoanaerobacter isolates reveals recombining, but differentiated, populations from geothermal springs of the Uzon Caldera, Kamchatka, Russia

    PubMed Central

    Wagner, Isaac D.; Varghese, Litty B.; Hemme, Christopher L.; Wiegel, Juergen

    2013-01-01

    Thermal environments have island-like characteristics and provide a unique opportunity to study population structure and diversity patterns of microbial taxa inhabiting these sites. Strains having ≥98% 16S rRNA gene sequence similarity to the obligately anaerobic Firmicutes Thermoanaerobacter uzonensis were isolated from seven geothermal springs, separated by up to 1600 m, within the Uzon Caldera (Kamchatka, Russian Far East). The intraspecies variation and spatial patterns of diversity for this taxon were assessed by multilocus sequence analysis (MLSA) of 106 strains. Analysis of eight protein-coding loci (gyrB, lepA, leuS, pyrG, recA, recG, rplB, and rpoB) revealed that all loci were polymorphic and that nucleotide substitutions were mostly synonymous. There were 148 variable nucleotide sites across 8003 bp concatenates of the protein-coding loci. While pairwise FST values indicated a small but significant level of genetic differentiation between most subpopulations, there was a negligible relationship between genetic divergence and spatial separation. Strains with the same allelic profile were only isolated from the same hot spring, occasionally from consecutive years, and single locus variant (SLV) sequence types were usually derived from the same spring. While recombination occurred, there was an “epidemic” population structure in which a particular T. uzonensis sequence type rose in frequency relative to the rest of the population. These results demonstrate spatial diversity patterns for an anaerobic bacterial species in a relative small geographic location and reinforce the view that terrestrial geothermal springs are excellent places to look for biogeographic diversity patterns regardless of the involved distances. PMID:23801987

  17. Molecular and iridescent feather reflectance data reveal recent genetic diversification and phenotypic differentiation in a cloud forest hummingbird.

    PubMed

    Ornelas, Juan Francisco; González, Clementina; Hernández-Baños, Blanca E; García-Moreno, Jaime

    2016-02-01

    The present day distribution and spatial genetic diversity of Mesoamerican biota reflects a long history of responses to habitat change. The hummingbird Lampornis amethystinus is distributed in northern Mesoamerica, with geographically disjunct populations. Based on sampling across the species range using mitochondrial DNA (mtDNA) sequences and nuclear microsatellites jointly analysed with phenotypic and climatic data, we (1) test whether the fragmented distribution is correlated with main evolutionary lineages, (2) assess body size and plumage color differentiation of populations in geographic isolation, and (3) evaluate a set of divergence scenarios and demographic patterns of the hummingbird populations. Analysis of genetic variation revealed four main groups: blue-throated populations (Sierra Madre del Sur); two groups of amethyst-throated populations (Trans-Mexican Volcanic Belt and Sierra Madre Oriental); and populations east of the Isthmus of Tehuantepec (IT) with males showing an amethyst throat. The most basal split is estimated to have originated in the Pleistocene, 2.39-0.57 million years ago (MYA), and corresponded to groups of populations separated by the IT. However, the estimated recent divergence time between blue- and amethyst-throated populations does not correspond to the 2-MY needed to be in isolation for substantial plumage divergence, likely because structurally iridescent colors are more malleable than others. Results of species distribution modeling and Approximate Bayesian Computation analysis fit a model of lineage divergence west of the Isthmus after the Last Glacial Maximum (LGM), and that the species' suitable habitat was disjunct during past and current conditions. These results challenge the generality of the contraction/expansion glacial model to cloud forest-interior species and urges management of cloud forest, a highly vulnerable ecosystem to climate change and currently facing destruction, to prevent further loss of genetic

  18. Analysis of SM22alpha-deficient mice reveals unanticipated insights into smooth muscle cell differentiation and function.

    PubMed

    Zhang, J C; Kim, S; Helmke, B P; Yu, W W; Du, K L; Lu, M M; Strobeck, M; Yu, Q; Parmacek, M S

    2001-02-01

    SM22alpha is a 22-kDa smooth muscle cell (SMC) lineage-restricted protein that physically associates with cytoskeletal actin filament bundles in contractile SMCs. To examine the function of SM22alpha, gene targeting was used to generate SM22alpha-deficient (SM22(-/-LacZ)) mice. The gene targeting strategy employed resulted in insertion of the bacterial lacZ reporter gene at the SM22alpha initiation codon, permitting precise analysis of the temporal and spatial pattern of SM22alpha transcriptional activation in the developing mouse. Northern and Western blot analyses confirmed that the gene targeting strategy resulted in a null mutation. Histological analysis of SM22(+/-LacZ) embryos revealed detectable beta-galactosidase activity in the unturned embryonic day 8.0 embryo in the layer of cells surrounding the paired dorsal aortae concomitant with its expression in the primitive heart tube, cephalic mesenchyme, and yolk sac vasculature. Subsequently, during postnatal development, beta-galactosidase activity was observed exclusively in arterial, venous, and visceral SMCs. SM22alpha-deficient mice are viable and fertile. Their blood pressure and heart rate do not differ significantly from their control SM22alpha(+/-) and SM22alpha(+/+) littermates. The vasculature and SMC-containing tissues of SM22alpha-deficient mice develop normally and appear to be histologically and ultrastructurally similar to those of their control littermates. Taken together, these data demonstrate that SM22alpha is not required for basal homeostatic functions mediated by vascular and visceral SMCs in the developing mouse. These data also suggest that signaling pathways that regulate SMC specification and differentiation from local mesenchyme are activated earlier in the angiogenic program than previously recognized.

  19. Proteome Analysis of Rice (Oryza sativa L.) Mutants Reveals Differentially Induced Proteins during Brown Planthopper (Nilaparvata lugens) Infestation

    PubMed Central

    Sangha, Jatinder Singh; Yolanda, H. Chen; Kaur, Jatinder; Khan, Wajahatullah; Abduljaleel, Zainularifeen; Alanazi, Mohammed S.; Mills, Aaron; Adalla, Candida B.; Bennett, John; Prithiviraj, Balakrishnan; Jahn, Gary C.; Leung, Hei

    2013-01-01

    Although rice resistance plays an important role in controlling the brown planthopper (BPH), Nilaparvata lugens, not all varieties have the same level of protection against BPH infestation. Understanding the molecular interactions in rice defense response is an important tool to help to reveal unexplained processes that underlie rice resistance to BPH. A proteomics approach was used to explore how wild type IR64 and near-isogenic rice mutants with gain and loss of resistance to BPH respond during infestation. A total of 65 proteins were found markedly altered in wild type IR64 during BPH infestation. Fifty-two proteins associated with 11 functional categories were identified using mass spectrometry. Protein abundance was less altered at 2 and 14 days after infestation (DAI) (T1, T2, respectively), whereas higher protein levels were observed at 28 DAI (T3). This trend diminished at 34 DAI (T4). Comparative analysis of IR64 with mutants showed 22 proteins that may be potentially associated with rice resistance to the brown planthopper (BPH). Ten proteins were altered in susceptible mutant (D1131) whereas abundance of 12 proteins including S-like RNase, Glyoxalase I, EFTu1 and Salt stress root protein “RS1” was differentially changed in resistant mutant (D518). S-like RNase was found in greater quantities in D518 after BPH infestation but remained unchanged in IR64 and decreased in D1131. Taken together, this study shows a noticeable level of protein abundance in the resistant mutant D518 compared to the susceptible mutant D1131 that may be involved in rendering enhanced level of resistance against BPH. PMID:23434671

  20. Kisspeptin Antagonists Reveal Kisspeptin 1 and Kisspeptin 2 Differential Regulation of Reproduction in the Teleost, Morone saxatilis.

    PubMed

    Zmora, Nilli; Stubblefield, John David; Wong, Ten-Tsao; Levavi-Sivan, Berta; Millar, Robert Peter; Zohar, Yonathan

    2015-09-01

    The importance of kisspeptin in regulating vertebrate reproduction has been well established, but the exact mechanism continues to unfold. Unlike mammals, many lower vertebrates possess a dual kisspeptin system, Kiss1 and Kiss2. To decipher the roles of the kisspeptins in fish, we identified two potential kisspeptin antagonists, pep 234 and pep 359, by screening analogs for their ability to inactivate striped bass Kiss1 and Kiss2 receptors expressed in COS7 cells. Pep 234 (a mammalian KISS1 antagonist) antagonizes Kiss1r signaling activated by Kiss1 and Kiss2, and pep 359 (a novel analog) antagonizes Kiss2 activation of both receptors. In vitro studies using brain slices demonstrated that only Kiss2 can upregulate the expression of the hypophysiotropic gnrh1, which was subsequently diminished by pep 234 and pep 359. In primary pituitary cell cultures, the two antagonists revealed a complex network of putative endogenous and exogenous regulation by kisspeptin. While both kisspeptins stimulate Fsh expression and secretion, Kiss2 predominately induces Lh secretion. Pep 234 and 359 treatment of spawning males hindered sperm production. This effect was accompanied with decreased brain gnrh1 and gnrh2 mRNA levels and peptide content in the pituitary, and increased levels of pituitary Lh, probably due to attenuation of Lh release. Strikingly, the mRNA levels of arginine-vasotocin, the neurons of which in the preoptic area coexpress kiss2r, were dramatically reduced by the antagonists. Our results demonstrate differential actions of Kiss1 and Kiss2 systems along the hypothalamic-pituitary axis and interactions with other neuropeptides, and further reinforce the importance of kisspeptin in the execution of spawning.

  1. Multilocus sequence analysis of Thermoanaerobacter isolates reveals recombining, but differentiated, populations from geothermal springs of the Uzon Caldera, Kamchatka, Russia.

    PubMed

    Wagner, Isaac D; Varghese, Litty B; Hemme, Christopher L; Wiegel, Juergen

    2013-01-01

    Thermal environments have island-like characteristics and provide a unique opportunity to study population structure and diversity patterns of microbial taxa inhabiting these sites. Strains having ≥98% 16S rRNA gene sequence similarity to the obligately anaerobic Firmicutes Thermoanaerobacter uzonensis were isolated from seven geothermal springs, separated by up to 1600 m, within the Uzon Caldera (Kamchatka, Russian Far East). The intraspecies variation and spatial patterns of diversity for this taxon were assessed by multilocus sequence analysis (MLSA) of 106 strains. Analysis of eight protein-coding loci (gyrB, lepA, leuS, pyrG, recA, recG, rplB, and rpoB) revealed that all loci were polymorphic and that nucleotide substitutions were mostly synonymous. There were 148 variable nucleotide sites across 8003 bp concatenates of the protein-coding loci. While pairwise F ST values indicated a small but significant level of genetic differentiation between most subpopulations, there was a negligible relationship between genetic divergence and spatial separation. Strains with the same allelic profile were only isolated from the same hot spring, occasionally from consecutive years, and single locus variant (SLV) sequence types were usually derived from the same spring. While recombination occurred, there was an "epidemic" population structure in which a particular T. uzonensis sequence type rose in frequency relative to the rest of the population. These results demonstrate spatial diversity patterns for an anaerobic bacterial species in a relative small geographic location and reinforce the view that terrestrial geothermal springs are excellent places to look for biogeographic diversity patterns regardless of the involved distances.

  2. Unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not promote human monocyte differentiation toward alternative macrophages

    SciTech Connect

    Bouhlel, Mohamed Amine; Brozek, John; Derudas, Bruno; Zawadzki, Christophe; Jude, Brigitte; Staels, Bart; Chinetti-Gbaguidi, Giulia

    2009-08-28

    Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPAR{gamma} promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPAR{beta}/{delta} in this process has been reported only in mice and no data are available for PPAR{alpha}. Here, we show that in contrast to PPAR{gamma}, expression of PPAR{alpha} and PPAR{beta}/{delta} overall does not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPAR{alpha} and PPAR{beta}/{delta} do not appear to modulate the alternative differentiation of human macrophages.

  3. miR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL

    PubMed Central

    Zhang, Bo-Wen; Cai, Han-Fang; Wei, Xue-Feng; Sun, Jia-Jie; Lan, Xian-Yong; Lei, Chu-Zhao; Lin, Feng-Peng; Qi, Xing-Lei; Plath, Martin; Chen, Hong

    2016-01-01

    MicroRNAs (miRNAs), a class of single stranded, small (~22 nucleotides), non-coding RNAs, play an important role in muscle development. We focused on the role of the miR-30-5p family during bovine muscle development from previous high-throughput sequencing results and analyzed their expression profiles. MHC and MyoG mRNAs expression as well as their proteins were suppressed in differentiated C2C12 cells, suggesting the importance of miR-30-5p in muscle development. MBNL, the candidate target of miR-30-5p, is an alternative splicing regulation factor. MBNL1 and MBNL3 have opposite effects on muscle differentiation. Our results confirmed that miR-30a-5p and miR-30e-5p repress the expression of MBNL1, MBNL2 and MBNL3, whereas miR-30b-5p inhibits MBNL1 and MBNL2 expression. This provides direct evidence that MBNL expression can be flexibly regulated by miR-30-5p. Previous studies showed that MBNL1 promotes exon inclusion of two muscle-related genes (Trim55 and INSR). Through RNA splicing studies, we found that miR-30-5p had an effect on their alternative splicing, which means miR-30-5p via MBNL1 could be integrated into muscle signaling pathways in which INSR or Trim55 are located. In conclusion, miR-30-5p could inhibit muscle cell differentiation and regulate the alternative splicing of Trim55 and INSR by targeting MBNL. These results promote the understanding of the function of miRNAs in muscle development. PMID:26840300

  4. Differential Reinforcement of Alternative Behavior and Demand Fading in the Treatment of Escape-Maintained Destructive Behavior.

    ERIC Educational Resources Information Center

    Piazza, Cathleen C.; And Others

    1996-01-01

    A study of an 11-year-old boy with autism and mild mental retardation found that his escape-maintained destructive behavior was reduced during instructional sequences with differential reinforcement of compliance, escape extinction without physical guidance, and demand fading. The procedure decreased destructive behaviors to near-zero levels and…

  5. Differential reinforcement of alternative behavior and demand fading in the treatment of escape-maintained destructive behavior.

    PubMed

    Piazza, C C; Moes, D R; Fisher, W W

    1996-01-01

    The escape-maintained destructive behavior of a boy with autism was reduced during instructional sequences with differential reinforcement of compliance (DRA), escape extinction without physical guidance, and demand fading. The procedure decreased destructive behaviors to near-zero levels and greatly increased compliance.

  6. Gender-related differential item functioning in DSM-IV/DSM-5-III (alternative model) diagnostic criteria for borderline personality disorder.

    PubMed

    Benson, Kathryn T; Donnellan, M Brent; Morey, Leslie C

    2017-01-01

    A number of studies have evaluated the possibility of bias in the diagnostic criteria in borderline personality disorder as an explanation of gender differences in prevalence. Previous studies have used both regression and latent trait approaches but the results have been inconsistent. The current study extended prior investigations in testing differential function of Borderline diagnostic criteria using both regression and latent-trait methods in the same sample, examining both Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM-IV) and DSM-5 alternative model criteria for borderline personality. Data were obtained from a national sample of 337 clinicians providing diagnostic information on 1 of their target patients. Chronic feelings of emptiness was the only criterion that demonstrated consistent evidence of potential differential functioning across methods and diagnostic models. Implications of these results for the conceptualization of borderline personality are discussed. (PsycINFO Database Record

  7. Alternative splicing of a viral mirtron differentially affects the expression of other microRNAs from its cluster and of the host transcript.

    PubMed

    Rasschaert, Perrine; Figueroa, Thomas; Dambrine, Ginette; Rasschaert, Denis; Laurent, Sylvie

    2016-12-01

    Interplay between alternative splicing and the Microprocessor may have differential effects on the expression of intronic miRNAs organized into clusters. We used a viral model - the LAT long non-coding RNA (LAT lncRNA) of Marek's disease oncogenic herpesvirus (MDV-1), which has the mdv1-miR-M8-M6-M7-M10 cluster embedded in its first intron - to assess the impact of splicing modifications on the biogenesis of each of the miRNAs from the cluster. Drosha silencing and alternative splicing of an extended exon 2 of the LAT lncRNA from a newly identified 3' splice site (SS) at the end of the second miRNA of the cluster showed that mdv1-miR-M6 was a 5'-tailed mirtron. We have thus identified the first 5'-tailed mirtron within a cluster of miRNAs for which alternative splicing is directly associated with differential expression of the other miRNAs of the cluster, with an increase in intronic mdv1-miR-M8 expression and a decrease in expression of the exonic mdv1-miR-M7, and indirectly associated with regulation of the host transcript. According to the alternative 3SS used for the host intron splicing, the mdv1-miR-M6 is processed as a mirtron by the spliceosome, dispatching the other miRNAs of the cluster into intron and exon, or as a canonical miRNA by the Microprocessor complex. The viral mdv1-miR-M6 mirtron is the first mirtron described that can also follow the canonical pathway.

  8. Comparison of differential pulse and alternating current polarography in the soft-modelling study of the complexation of Cd(II) by the fragment Cys-Gly and by the phytochelatin (gamma-Glu-Cys)2Gly.

    PubMed

    Garrigosa, Anna Maria; Ariño, Cristina; Díaz-Cruz, José Manuel; Esteban, Miquel

    2008-07-01

    A comparison of a differential pulse polarographic with a phase sensitive alternating current polarographic study of the Cd-Cys-Gly and Cd-PC(2) systems [PC(2) being a phytochelatin of general structure (gamma-Glu-Cys)(n)-Gly, with n = 2] has been performed. The chemometric multivariate curve resolution method with alternating least squares was applied in the experimental data analysis. The results obtained by both polarographic techniques have made it possible to find out the formation sequences of the complexes and their final stoichiometries. The alternating current polarograms compared with the differential pulse ones show some differences (a new signal and an important shift of peak potentials), which anyway are consistent with some of the conclusions obtained by differential pulse polarography. This fact implies that although the alternating current polarography results need some corrections before data treatment, they provide extra information that complements the conclusions achieved by differential pulse polarography.

  9. Differential Gene Expression by Lactobacillus plantarum WCFS1 in Response to Phenolic Compounds Reveals New Genes Involved in Tannin Degradation.

    PubMed

    Reverón, Inés; Jiménez, Natalia; Curiel, José Antonio; Peñas, Elena; López de Felipe, Félix; de Las Rivas, Blanca; Muñoz, Rosario

    2017-04-01

    Lactobacillus plantarum is a lactic acid bacterium that can degrade food tannins by the successive action of tannase and gallate decarboxylase enzymes. In the L. plantarum genome, the gene encoding the catalytic subunit of gallate decarboxylase (lpdC, or lp_2945) is only 6.5 kb distant from the gene encoding inducible tannase (L. plantarumtanB [tanBLp ], or lp_2956). This genomic context suggests concomitant activity and regulation of both enzymatic activities. Reverse transcription analysis revealed that subunits B (lpdB, or lp_0271) and D (lpdD, or lp_0272) of the gallate decarboxylase are cotranscribed, whereas subunit C (lpdC, or lp_2945) is cotranscribed with a gene encoding a transport protein (gacP, or lp_2943). In contrast, the tannase gene is transcribed as a monocistronic mRNA. Investigation of knockout mutations of genes located in this chromosomal region indicated that only mutants of the gallate decarboxylase (subunits B and C), tannase, GacP transport protein, and TanR transcriptional regulator (lp_2942) genes exhibited altered tannin metabolism. The expression profile of genes involved in tannin metabolism was also analyzed in these mutants in the presence of methyl gallate and gallic acid. It is noteworthy that inactivation of tanR suppresses the induction of all genes overexpressed in the presence of methyl gallate and gallic acid. This transcriptional regulator was also induced in the presence of other phenolic compounds, such as kaempferol and myricetin. This study complements the catalog of L. plantarum expression profiles responsive to phenolic compounds, which enable this bacterium to adapt to a plant food environment.IMPORTANCELactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables when tannins are present. L. plantarum strains degrade tannins to the less-toxic pyrogallol by the successive action of tannase and gallate decarboxylase enzymes. The genes encoding these enzymes are located close to each

  10. Topographic changes due to the 2008 Mw 7.9 Wenchuan earthquake as revealed by the differential DEM method

    NASA Astrophysics Data System (ADS)

    Ren, Zhikun; Zhang, Zhuqi; Dai, Fuchu; Yin, Jinhui; Zhang, Huiping

    2014-07-01

    Landscape evolution in active orogenic regions is inevitably affected by the repeated strong earthquakes triggered by the corresponding active faults. However, the lack of adequate methods for the documentation and monitoring of mountain-building processes has resulted in a shortage of quantitative estimates of orogenic and eroded volumes. A strong earthquake and its associated co-seismic landslides represent a sudden pulse in landscape evolution in tectonically active areas. The 2008 Mw 7.9 Wenchuan earthquake dramatically modified the topography of the Longmen Shan region. Based on topographic data before the earthquake and stereo pairs of post-earthquake remote sensing imagery, we derived pre- and post-earthquake DEMs (digital elevation models) of the three regions along the Longmen Shan Thrust Belt. By comparing the geomorphic features before and after the earthquake, we find that the Wenchuan earthquake smoothed the steep relief and caused a co-seismic uplift of the Longmen Shan region. The medium-relief regions increased; however, the high-relief regions decreased, indicating that the local relief is controlled by repeated strong earthquakes. The changed slope aspect indicates that the formation and modification of the east- and west-facing slopes are controlled by tectonic events in the Longmen Shan region, which might be associated with the regional stress field. However, the unchanged aspects of other slopes might be controlled by long-term erosion rather than tectonic events. The topographic changes, landslide volume and co-seismic uplift indicate that the greatest seismically induced denudation occurred in association with a thrust faulting mechanism and low-angle fault geometry. Our findings reveal that the local relief has been shaped by the localized, seismically induced high rate of denudation within the plateau margins, and that the formation of local relief is also related to tectonic events, especially the events that have occurred on low

  11. Fibrillin binds calcium and is coded by cDNAs that reveal a multidomain structure and alternatively spliced exons at the 5[prime] end

    SciTech Connect

    Corson, G.M.; Chalberg, S.C.; Charbonneau, N.L.; Sakai, L.Y. ); Dietz, H.C. )

    1993-08-01

    Fibrillin is an important structural protein of the extracellular matrix. It is a large cysteine-rich glycoprotein with extensive intrachain disulfide bonds, likely contributed by multiple EGF-like repeats. The authors have previously published 6.9 kb of FBN1 cDNA sequence. FBN1 cDNA clones that extend the sequence 3089 bp in the 5[prime] direction are described in this report. The deduced primary structure suggests that fibrillin in composed of multiple domains. The most predominant features the presence of 43 calcium binding EGF-like repeats. They demonstrate here that fibrillin molecules bind calcium. In addition, three alternatively spliced exons at the 5[prime] end are described. Analysis of 5.8 kb of surrounding genomic sequence revealed a 1.8-kb CpG island spanning the alternatively spliced exons and the next downstream exon. Since FBN1 is the gene responsible for Marfan syndrome, the information presented here will be useful in identifying new mutations and in understanding the function of fibrillin in the pathogenesis of the disease. 42 refs., 7 figs.

  12. Energy dispersive x-ray diffractometry as a tool alternative to differential scanning calorimetry for investigating polymer phase transitions

    NASA Astrophysics Data System (ADS)

    Rossi-Albertini, V.; Isopo, A.; Caminiti, R.; Tentolini, U.

    2002-02-01

    Recently, a technique based on energy dispersive x-ray diffraction has been proposed to follow the polymer phase transitions. However, the potentialities of this method were not clear, as well as the experimental conditions in which it is more convenient than differential scanning calorimetry, generally used for the same purpose. In the present letter, the answer to this question is provided. It is shown that the two methods are complementary, rather than equivalent, the heating rate being the relevant parameter to establish which is preferable. The demonstration of this statement is given through the observation of the complex thermal properties of a reference sample studied in both ways at progressively lower heating rates. The connection between such unusual application of x-ray diffraction and the differential scanning calorimetry is discussed in terms of the two possible definitions of entropy.

  13. Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

    PubMed

    Maya-Espinosa, Guadalupe; Collazo-Navarrete, Omar; Millán-Aldaco, Diana; Palomero-Rivero, Marcela; Guerrero-Flores, Gilda; Drucker-Colín, René; Covarrubias, Luis; Guerra-Crespo, Magdalena

    2015-02-01

    A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies.

  14. Evaluation of magnesium alloys with alternative surface finishing for the proliferation and chondro-differentiation of human mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Trinidad, J.; Arruebarrena, G.; Sáenz De Argandoña, E.; Ruiz De Eguino, G.; Infante, A.; Rodríguez, C. I.

    2010-11-01

    Articular cartilage has little capacity for self-repair. As a result, continuous mechanical stress can lead to the degradation of articular cartilage, culminating in progressive damage and joint degeneration. Tissue engineering has arisen as a promising therapeutic approach to cartilage repair. Magnesium alloys are one of the most important metallic biomaterials emerging in this area due to their biocompatibility, bio-absorbability and especially to their mechanical properties. These properties make magnesium alloys a promising biomaterial in the regeneration of cartilage tissue. Objective. This study was undertaken to analyze the influence of surface characteristics of magnesium alloys in the adhesion, proliferation and differentiation of human mesenchymal stem cells (MSCs). Methods. Two commercial magnesium alloys (AZ31B and ZM21) were subjected to different treatments in order to obtain four different surfaces in each alloy. Human MSCs were seeded into the magnesium alloys and analyzed for their proliferation and chondrogenesis differentiation ability. Results. Human MSCs showed a greater proliferation and chondro-differentiation when cultured in the ZM21 magnesium alloy with a surface finishing of fine sanding, polishing, and etching.

  15. New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

    PubMed Central

    2013-01-01

    Background Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. Results From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Conclusions Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. PMID:24148232

  16. Alternative S2 hinge regions of the myosin rod differentially affect muscle function, myofibril dimensions and myosin tail length

    PubMed Central

    Suggs, Jennifer A.; Cammarato, Anthony; Kronert, William A.; Nikkhoy, Massoud; Dambacher, Corey M.; Megighian, Aram; Bernstein, Sanford I.

    2007-01-01

    Muscle myosin heavy chain (MHC) rod domains intertwine to form alpha-helical coiled-coil dimers; these subsequently multimerize into thick filaments via electrostatic interactions. The subfragment 2/light meromyosin “hinge” region of the MHC rod, located in the C-terminal third of heavy meromyosin, may form a less stable coiled-coil than flanking regions. Partial “melting” of this region has been proposed to result in a helix to random-coil transition. A portion of the Drosophila melanogaster MHC hinge is encoded by mutually exclusive alternative exons 15a and 15b, the use of which correlates with fast (hinge A) or slow (hinge B) muscle physiological properties. To test the functional significance of alternative hinge regions, we constructed transgenic fly lines in which fast muscle isovariant hinge A was switched for slow muscle hinge B in the MHC isoforms of indirect flight and jump muscles. Substitution of the slow muscle hinge B impaired flight ability, increased sarcomere lengths by approximately 13% and resulted in minor disruption to indirect flight muscle sarcomeric structure compared with a transgenic control. With age, residual flight ability decreased rapidly and myofibrils developed peripheral defects. Computational analysis indicates that hinge B has a greater coiled-coil propensity and thus reduced flexibility compared to hinge A. Intriguingly, the MHC rod with hinge B was ~5 nm longer than myosin with hinge A, consistent with the more rigid coiled-coil conformation predicted for hinge B. Our study demonstrates that hinge B cannot functionally substitute for hinge A in fast muscle types, likely as a result of differences in the molecular structure of the rod, subtle changes in myofibril structure and decreased ability to maintain sarcomere structure in indirect flight muscle myofibrils. Thus alternative hinges are important in dictating the distinct functional properties of myosin isoforms and the muscles in which they are expressed. PMID

  17. Comprehensive Transcriptome Profiling Reveals Long Noncoding RNA Expression and Alternative Splicing Regulation during Fruit Development and Ripening in Kiwifruit (Actinidia chinensis)

    PubMed Central

    Tang, Wei; Zheng, Yi; Dong, Jing; Yu, Jia; Yue, Junyang; Liu, Fangfang; Guo, Xiuhong; Huang, Shengxiong; Wisniewski, Michael; Sun, Jiaqi; Niu, Xiangli; Ding, Jian; Liu, Jia; Fei, Zhangjun; Liu, Yongsheng

    2016-01-01

    Genomic and transcriptomic data on kiwifruit (Actinidia chinensis) in public databases are very limited despite its nutritional and economic value. Previously, we have constructed and sequenced nine fruit RNA-Seq libraries of A. chinensis “Hongyang” at immature, mature, and postharvest ripening stages of fruit development, and generated over 66.2 million paired-end and 24.4 million single-end reads. From this dataset, here we have identified 7051 long noncoding RNAs (lncRNAs), 29,327 alternative splicing (AS) events and 2980 novel protein-coding genes that were not annotated in the draft genome of “Hongyang.” AS events were demonstrated in genes involved in the synthesis of nutritional metabolites in fruit, such as ascorbic acids, carotenoids, anthocyanins, and chlorophylls, and also in genes in the ethylene signaling pathway, which plays an indispensable role in fruit ripening. Additionally, transcriptome profiles and the contents of sugars, organic and main amino acids were compared between immature, mature, and postharvest ripening stages in kiwifruits. A total of 5931 differentially expressed genes were identified, including those associated with the metabolism of sugar, organic acid, and main amino acids. The data generated in this study provide a foundation for further studies of fruit development and ripening in kiwifruit, and identify candidate genes and regulatory elements that could serve as targets for improving important agronomic traits through marker assisted breeding and biotechnology. PMID:27594858

  18. Whole-Exome Sequencing Identifies Loci Associated with Blood Cell Traits and Reveals a Role for Alternative GFI1B Splice Variants in Human Hematopoiesis.

    PubMed

    Polfus, Linda M; Khajuria, Rajiv K; Schick, Ursula M; Pankratz, Nathan; Pazoki, Raha; Brody, Jennifer A; Chen, Ming-Huei; Auer, Paul L; Floyd, James S; Huang, Jie; Lange, Leslie; van Rooij, Frank J A; Gibbs, Richard A; Metcalf, Ginger; Muzny, Donna; Veeraraghavan, Narayanan; Walter, Klaudia; Chen, Lu; Yanek, Lisa; Becker, Lewis C; Peloso, Gina M; Wakabayashi, Aoi; Kals, Mart; Metspalu, Andres; Esko, Tõnu; Fox, Keolu; Wallace, Robert; Franceshini, Nora; Matijevic, Nena; Rice, Kenneth M; Bartz, Traci M; Lyytikäinen, Leo-Pekka; Kähönen, Mika; Lehtimäki, Terho; Raitakari, Olli T; Li-Gao, Ruifang; Mook-Kanamori, Dennis O; Lettre, Guillaume; van Duijn, Cornelia M; Franco, Oscar H; Rich, Stephen S; Rivadeneira, Fernando; Hofman, Albert; Uitterlinden, André G; Wilson, James G; Psaty, Bruce M; Soranzo, Nicole; Dehghan, Abbas; Boerwinkle, Eric; Zhang, Xiaoling; Johnson, Andrew D; O'Donnell, Christopher J; Johnsen, Jill M; Reiner, Alexander P; Ganesh, Santhi K; Sankaran, Vijay G

    2016-08-04

    Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0.33, discovery + replication p = 6.38 × 10(-10)) and a rare synonymous variant in GFI1B (rs150813342, MAF = 0.009, discovery + replication p = 1.79 × 10(-27)). By performing CRISPR/Cas9 genome editing in hematopoietic cell lines and follow-up targeted knockdown experiments in primary human hematopoietic stem and progenitor cells, we demonstrate an alternative splicing mechanism by which the GFI1B rs150813342 variant suppresses formation of a GFI1B isoform that preferentially promotes megakaryocyte differentiation and platelet production. These results demonstrate how unbiased studies of natural variation in blood cell traits can provide insight into the regulation of human hematopoiesis.

  19. Comprehensive Transcriptome Profiling Reveals Long Noncoding RNA Expression and Alternative Splicing Regulation during Fruit Development and Ripening in Kiwifruit (Actinidia chinensis).

    PubMed

    Tang, Wei; Zheng, Yi; Dong, Jing; Yu, Jia; Yue, Junyang; Liu, Fangfang; Guo, Xiuhong; Huang, Shengxiong; Wisniewski, Michael; Sun, Jiaqi; Niu, Xiangli; Ding, Jian; Liu, Jia; Fei, Zhangjun; Liu, Yongsheng

    2016-01-01

    Genomic and transcriptomic data on kiwifruit (Actinidia chinensis) in public databases are very limited despite its nutritional and economic value. Previously, we have constructed and sequenced nine fruit RNA-Seq libraries of A. chinensis "Hongyang" at immature, mature, and postharvest ripening stages of fruit development, and generated over 66.2 million paired-end and 24.4 million single-end reads. From this dataset, here we have identified 7051 long noncoding RNAs (lncRNAs), 29,327 alternative splicing (AS) events and 2980 novel protein-coding genes that were not annotated in the draft genome of "Hongyang." AS events were demonstrated in genes involved in the synthesis of nutritional metabolites in fruit, such as ascorbic acids, carotenoids, anthocyanins, and chlorophylls, and also in genes in the ethylene signaling pathway, which plays an indispensable role in fruit ripening. Additionally, transcriptome profiles and the contents of sugars, organic and main amino acids were compared between immature, mature, and postharvest ripening stages in kiwifruits. A total of 5931 differentially expressed genes were identified, including those associated with the metabolism of sugar, organic acid, and main amino acids. The data generated in this study provide a foundation for further studies of fruit development and ripening in kiwifruit, and identify candidate genes and regulatory elements that could serve as targets for improving important agronomic traits through marker assisted breeding and biotechnology.

  20. Keratinocytes exposed to ultraviolet radiation reveal three down-regulated genes with potential function in differentiation and cell cycle control.

    PubMed

    Pötter, T; Göhde, W; Wedemeyer, N; Köhnlein, W

    2000-08-01

    The incidence of skin cancer is increasing in epidemic proportion. Although solar UV radiation is known to be the major risk factor, much information is lacking about the molecular mechanisms leading to skin cancer. To gain a deeper insight into these mechanisms, we have examined cells of a human keratinocyte cell line (HaCat) after exposure to 0.16 minimal erythema doses of UVB radiation. This dose led to an S-phase delay that was reversible 22 h postirradiation. To examine gene expression 10 h after UV irradiation, a nonradioactive differential display was employed. Three genes were identified as being down-regulated significantly. The first encodes for topoisomerase-IIbeta-binding protein 1 (expression level 5% 6 h after irradiation). This protein is associated with human topoisomerase IIbeta and appears to be necessary for DNA replication during the onset of S phase. The second gene product has previously been reported to be involved in differentiation and is therefore known as differentiation-dependent A4 protein (28% 8 h after irradiation). The third gene is XPO1 (also known as CRM1) (5% 8 h after irradiation), whose protein is involved in nuclear export of mRNA molecules. Differential expression of these genes after UV irradiation has not been reported. Because of their potential involvement in cell cycle control and differentiation, these proteins could be important for understanding the reaction of keratinocytes after exposure to UV radiation.

  1. Differential expression and alternative splicing of rice sulphate transporter family members regulate sulphur status during plant growth, development and stress conditions.

    PubMed

    Kumar, Smita; Asif, Mehar Hasan; Chakrabarty, Debasis; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar

    2011-06-01

    Sulphur, an essential nutrient required for plant growth and development, is mainly taken up by the plants as inorganic sulphate from the soil and assimilated into the sulphur reductive pathway. The uptake and transport of sulphate in plants is carried out by transporters encoded by the sulphate transporter gene family. Plant sulphate transporters have been classified with respect to their protein sequences, kinetic properties and tissue-specific localization in Arabidopsis. Though sulphate transporter genes from few other plants have also been characterized, no detailed study with respect to the structure and expression of this family from rice has been carried out. Here, we present genome-wide identification, structural and expression analyses of the rice sulphate transporter gene family. Our analysis using microarray data and MPSS database suggests that 14 rice sulphate transporters are differentially expressed during growth and development in various tissues and during biotic and abiotic stresses. Our analysis also suggests differential accumulation of splice variants of OsSultr1;1 and OsSultr4;1 transcripts during these processes. Apart from known spliced variants, we report an unusual alternative splicing of OsSultr1;1 transcript related to sulphur supply in growth medium and during stress response. Taken together, our study suggests that differential expression and alternative splicing of members of the sulphate transporter family plays an important role in regulating cellular sulphur status required for growth and development and during stress conditions. These findings significantly advance our understanding of the posttranscriptional regulatory mechanisms operating to regulate sulphur demand by the plant.

  2. Lagrangian Descriptors for Stochastic Differential Equations: A Tool for Revealing the Phase Portrait of Stochastic Dynamical Systems

    NASA Astrophysics Data System (ADS)

    Balibrea-Iniesta, Francisco; Lopesino, Carlos; Wiggins, Stephen; Mancho, Ana M.

    2016-12-01

    In this paper, we introduce a new technique for depicting the phase portrait of stochastic differential equations. Following previous work for deterministic systems, we represent the phase space by means of a generalization of the method of Lagrangian descriptors to stochastic differential equations. Analogously to the deterministic differential equations setting, the Lagrangian descriptors graphically provide the distinguished trajectories and hyperbolic structures arising within the stochastic dynamics, such as random fixed points and their stable and unstable manifolds. We analyze the sense in which structures form barriers to transport in stochastic systems. We apply the method to several benchmark examples where the deterministic phase space structures are well-understood. In particular, we apply our method to the noisy saddle, the stochastically forced Duffing equation, and the stochastic double gyre model that is a benchmark for analyzing fluid transport.

  3. Application of low-frequency alternating current electric fields via interdigitated electrodes: effects on cellular viability, cytoplasmic calcium, and osteogenic differentiation of human adipose-derived stem cells.

    PubMed

    McCullen, Seth D; McQuilling, John P; Grossfeld, Robert M; Lubischer, Jane L; Clarke, Laura I; Loboa, Elizabeth G

    2010-12-01

    Electric stimulation is known to initiate signaling pathways and provides a technique to enhance osteogenic differentiation of stem and/or progenitor cells. There are a variety of in vitro stimulation devices to apply electric fields to such cells. Herein, we describe and highlight the use of interdigitated electrodes to characterize signaling pathways and the effect of electric fields on the proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). The advantage of the interdigitated electrode configuration is that cells can be easily imaged during short-term (acute) stimulation, and this identical configuration can be utilized for long-term (chronic) studies. Acute exposure of hASCs to alternating current (AC) sinusoidal electric fields of 1 Hz induced a dose-dependent increase in cytoplasmic calcium in response to electric field magnitude, as observed by fluorescence microscopy. hASCs that were chronically exposed to AC electric field treatment of 1 V/cm (4 h/day for 14 days, cultured in the osteogenic differentiation medium containing dexamethasone, ascorbic acid, and β-glycerol phosphate) displayed a significant increase in mineral deposition relative to unstimulated controls. This is the first study to evaluate the effects of sinusoidal AC electric fields on hASCs and to demonstrate that acute and chronic electric field exposure can significantly increase intracellular calcium signaling and the deposition of accreted calcium under osteogenic stimulation, respectively.

  4. Transcriptome meta-analysis reveals common differential and global gene expression profiles in cystic fibrosis and other respiratory disorders and identifies CFTR regulators.

    PubMed

    Clarke, Luka A; Botelho, Hugo M; Sousa, Lisete; Falcao, Andre O; Amaral, Margarida D

    2015-11-01

    A meta-analysis of 13 independent microarray data sets was performed and gene expression profiles from cystic fibrosis (CF), similar disorders (COPD: chronic obstructive pulmonary disease, IPF: idiopathic pulmonary fibrosis, asthma), environmental conditions (smoking, epithelial injury), related cellular processes (epithelial differentiation/regeneration), and non-respiratory "control" conditions (schizophrenia, dieting), were compared. Similarity among differentially expressed (DE) gene lists was assessed using a permutation test, and a clustergram was constructed, identifying common gene markers. Global gene expression values were standardized using a novel approach, revealing that similarities between independent data sets run deeper than shared DE genes. Correlation of gene expression values identified putative gene regulators of the CF transmembrane conductance regulator (CFTR) gene, of potential therapeutic significance. Our study provides a novel perspective on CF epithelial gene expression in the context of other lung disorders and conditions, and highlights the contribution of differentiation/EMT and injury to gene signatures of respiratory disease.

  5. Genomic Data Reveal Toxoplasma gondii Differentiation Mutants Are Also Impaired with Respect to Switching into a Novel Extracellular Tachyzoite State

    PubMed Central

    Lescault, Pamela J.; Thompson, Ann B.; Patil, Veerupaxagouda; Lirussi, Dario; Burton, Amanda; Margarit, Juan; Bond, Jeffrey; Matrajt, Mariana

    2010-01-01

    Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states – genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage. PMID:21209930

  6. A Conditional Knockout Mouse Model Reveals a Critical Role of PKD1 in Osteoblast Differentiation and Bone Development

    PubMed Central

    Li, Shao; Xu, Wanfu; Xing, Zhe; Qian, Jiabi; Chen, Liping; Gu, Ruonan; Guo, Wenjing; Lai, Xiaoju; Zhao, Wanlu; Li, Songyu; Wang, Yaodong; Wang, Q. Jane; Deng, Fan

    2017-01-01

    The protein kinase D family of serine/threonine kinases, particularly PKD1, has been implicated in the regulation of a complex array of fundamental biological processes. However, its function and mechanism underlying PKD1-mediated the bone development and osteoblast differentiation are not fully understood. Here we demonstrate that loss of PKD1 function led to impaired bone development and osteoblast differentiation through STAT3 and p38 MAPK signaling using in vitro and in vivo bone-specific conditional PKD1-knockout (PKD1-KO) mice models. These mice developed markedly craniofacial dysplasia, scapula dysplasia, long bone length shortage and body weight decrease compared with wild-type littermates. Moreover, deletion of PKD1 in vivo reduced trabecular development and activity of osteoblast development, confirmed by Micro-CT and histological staining as well as expression of osteoblastic marker (OPN, Runx2 and OSX). Mechanistically, loss of PKD1 mediated the downregulation of osteoblast markers and impaired osteoblast differentiation through STAT3 and p38 MAPK signaling pathways. Taken together, these results demonstrated that PKD1 contributes to the osteoblast differentiation and bone development via elevation of osteoblast markers through activation of STAT3 and p38 MAPK signaling pathways. PMID:28084409

  7. Systems Chemo-Biology and Transcriptomic Meta-Analysis Reveal the Molecular Roles of Bioactive Lipids in Cardiomyocyte Differentiation.

    PubMed

    de Faria Poloni, Joice; Bonatto, Diego

    2015-09-01

    Lipids, which are essential constituents of biological membranes, play structural and functional roles in the cell. In recent years, certain lipids have been identified as regulatory signaling molecules and have been termed "bioactive lipids". Subsequently, the importance of bioactive lipids in stem cell differentiation and cardiogenesis has gained increasing recognition. Therefore, the aim of this study was to identify the biological processes underlying murine cardiac differentiation and the mechanisms by which bioactive lipids affect these processes. For this purpose, a transcriptomic meta-analysis of microarray and RNA-seq data from murine stem cells undergoing cardiogenic differentiation was performed. The differentially expressed genes identified via this meta-analysis, as well as bioactive lipids, were evaluated using systems chemo-biology tools. These data indicated that bioactive lipids are associated with the regulation of cell motility, cell adhesion, cytoskeletal rearrangement, and gene expression. Moreover, bioactive lipids integrate the signaling pathways involved in cell migration, the secretion and remodeling of extracellular matrix components, and the establishment of the cardiac phenotype. In conclusion, this study provides new insights into the contribution of bioactive lipids to the induction of cellular responses to various stimuli, which may originate from the extracellular environment and morphogens, and the manner in which this contribution directly affects murine heart morphogenesis.

  8. cDNA-AFLP analysis reveals differential gene expression in response to salt stress in foxtail millet (Setaria italica L.).

    PubMed

    Jayaraman, Ananthi; Puranik, Swati; Rai, Neeraj Kumar; Vidapu, Sudhakar; Sahu, Pranav Pankaj; Lata, Charu; Prasad, Manoj

    2008-11-01

    Plant growth and productivity are affected by various abiotic stresses such as heat, drought, cold, salinity, etc. The mechanism of salt tolerance is one of the most important subjects in plant science as salt stress decreases worldwide agricultural production. In our present study we used cDNA-AFLP technique to compare gene expression profiles of a salt tolerant and a salt-sensitive cultivar of foxtail millet (Seteria italica) in response to salt stress to identify early responsive differentially expressed transcripts accumulated upon salt stress and validate the obtained result through quantitative real-time PCR (qRT-PCR). The expression profile was compared between a salt tolerant (Prasad) and susceptible variety (Lepakshi) of foxtail millet in both control condition (L0 and P0) and after 1 h (L1 and P1) of salt stress. We identified 90 transcript-derived fragments (TDFs) that are differentially expressed, out of which 86 TDFs were classified on the basis of their either complete presence or absence (qualitative variants) and 4 on differential expression pattern levels (quantitative variants) in the two varieties. Finally, we identified 27 non-redundant differentially expressed cDNAs that are unique to salt tolerant variety which represent different groups of genes involved in metabolism, cellular transport, cell signaling, transcriptional regulation, mRNA splicing, seed development and storage, etc. The expression patterns of seven out of nine such genes showed a significant increase of differential expression in tolerant variety after 1 h of salt stress in comparison to salt-sensitive variety as analyzed by qRT-PCR. The direct and indirect relationship of identified TDFs with salinity tolerance mechanism is discussed.

  9. A Genome-Wide Test of the Differential Susceptibility Hypothesis Reveals a Genetic Predictor of Differential Response to Psychological Treatments for Child Anxiety Disorders

    PubMed Central

    Keers, Robert; Coleman, Jonathan R.I.; Lester, Kathryn J.; Roberts, Susanna; Breen, Gerome; Thastum, Mikael; Bögels, Susan; Schneider, Silvia; Heiervang, Einar; Meiser-Stedman, Richard; Nauta, Maaike; Creswell, Cathy; Thirlwall, Kerstin; Rapee, Ronald M.; Hudson, Jennifer L.; Lewis, Cathryn; Plomin, Robert; Eley, Thalia C.

    2016-01-01

    Background The differential susceptibly hypothesis suggests that certain genetic variants moderate the effects of both negative and positive environments on mental health and may therefore be important predictors of response to psychological treatments. Nevertheless, the identification of such variants has so far been limited to preselected candidate genes. In this study we extended the differential susceptibility hypothesis from a candidate gene to a genome-wide approach to test whether a polygenic score of environmental sensitivity predicted response to cognitive behavioural therapy (CBT) in children with anxiety disorders. Methods We identified variants associated with environmental sensitivity using a novel method in which within-pair variability in emotional problems in 1,026 monozygotic twin pairs was examined as a function of the pairs' genotype. We created a polygenic score of environmental sensitivity based on the whole-genome findings and tested the score as a moderator of parenting on emotional problems in 1,406 children and response to individual, group and brief parent-led CBT in 973 children with anxiety disorders. Results The polygenic score significantly moderated the effects of parenting on emotional problems and the effects of treatment. Individuals with a high score responded significantly better to individual CBT than group CBT or brief parent-led CBT (remission rates: 70.9, 55.5 and 41.6%, respectively). Conclusions Pending successful replication, our results should be considered exploratory. Nevertheless, if replicated, they suggest that individuals with the greatest environmental sensitivity may be more likely to develop emotional problems in adverse environments but also benefit more from the most intensive types of treatment. PMID:27043157

  10. Transcriptomic meta-analysis reveals up-regulation of gene expression functional in osteoclast differentiation in human septic shock.

    PubMed

    Mukhopadhyay, Samanwoy; Thatoi, Pravat K; Pandey, Abhay D; Das, Bidyut K; Ravindran, Balachandran; Bhattacharjee, Samsiddhi; Mohapatra, Saroj K

    2017-01-01

    Septic shock is a major medical problem with high morbidity and mortality and incompletely understood biology. Integration of multiple data sets into a single analysis framework empowers discovery of new knowledge about the condition that may have been missed by individual analysis of each of these datasets. Electronic search was performed on medical literature and gene expression databases for selection of transcriptomic studies done in circulating leukocytes from human subjects suffering from septic shock. Gene-level meta-analysis was conducted on the six selected studies to identify the genes consistently differentially expressed in septic shock. This was followed by pathway-level analysis using three different algorithms (ORA, GSEA, SPIA). The identified up-regulated pathway, Osteoclast differentiation pathway (hsa04380) was validated in two independent cohorts. Of the pathway, 25 key genes were selected that serve as an expression signature of Septic Shock.

  11. Transcriptomic meta-analysis reveals up-regulation of gene expression functional in osteoclast differentiation in human septic shock

    PubMed Central

    Mukhopadhyay, Samanwoy; Thatoi, Pravat K.; Pandey, Abhay D.; Das, Bidyut K.; Ravindran, Balachandran; Bhattacharjee, Samsiddhi; Mohapatra, Saroj K.

    2017-01-01

    Septic shock is a major medical problem with high morbidity and mortality and incompletely understood biology. Integration of multiple data sets into a single analysis framework empowers discovery of new knowledge about the condition that may have been missed by individual analysis of each of these datasets. Electronic search was performed on medical literature and gene expression databases for selection of transcriptomic studies done in circulating leukocytes from human subjects suffering from septic shock. Gene-level meta-analysis was conducted on the six selected studies to identify the genes consistently differentially expressed in septic shock. This was followed by pathway-level analysis using three different algorithms (ORA, GSEA, SPIA). The identified up-regulated pathway, Osteoclast differentiation pathway (hsa04380) was validated in two independent cohorts. Of the pathway, 25 key genes were selected that serve as an expression signature of Septic Shock. PMID:28199355

  12. Differential Gene Expression Reveals Mitochondrial Dysfunction in an Imprinting Center Deletion Mouse Model of Prader-Willi Syndrome

    PubMed Central

    Fan, Weiwei; Coskun, Pinar E.; Nalbandian, Angèle; Knoblach, Susan; Resnick, James L.; Hoffman, Eric; Wallace, Douglas C.; Kimonis, Virginia E.

    2013-01-01

    Prader-Willi syndrome (PWS) is a genetic disorder caused by deficiency of imprinted gene expression from the paternal chromosome 15q11-15q13 and clinically characterized by neonatal hypotonia, short stature, cognitive impairment, hypogonadism, hyperphagia, morbid obesity and diabetes. Previous clinical studies suggest that a defect in energy metabolism may be involved in the pathogenesis of PWS. We focused our attention on the genes associated with energy metabolism and found that there were 95 and 66 mitochondrial genes differentially expressed in PWS muscle and brain, respectively. Assessment of enzyme activities of mitochondrial oxidative phosphorylation (OXPHOS) complexes in the brain, heart, liver and muscle were assessed. We found the enzyme activities of the cardiac mitochondrial complexes II+III were upregulated in the imprinting center deletion (PWS-IC) mice compared to the wild type littermates. These studies suggest that differential gene expression, especially of the mitochondrial genes may contribute to the pathophysiology of PWS. PMID:24127921

  13. Atypical squamous cells in the urine revealing endometrioid adenocarcinoma of the endometrium with squamous cell differentiation: a case report.

    PubMed

    Wang, Yinong; Otis, Christopher N; Florence, Roxanne R

    2015-01-01

    Urine cytology is mainly used to detect urothelial carcinoma (UC), especially for high-grade lesions including urothelial carcinoma in situ. Benign squamous cells are often seen in the urine specimens of women, they are either exfoliated from the trigone area of the bladder, the urethra, or the cervicovaginal region. However, abnormal squamous cells in the urine raise concerns of abnormalities of the urinary tract and cervicovaginal area which range from squamous metaplasia of the urothelium, a cervicovaginal squamous intraepithelial lesion, condyloma acuminatum of the bladder, UC with squamous differentiation, and squamous cell carcinoma. We present here a unique case of atypical squamous cells (ASCs) in the urine subsequently leading to the diagnosis of endometrioid adenocarcinoma of the endometrium with squamous differentiation. The presence of ASCs in voided urine is a rare finding that may indicate an underlying malignancy. Careful evaluation of squamous cells in the urine is an important part of our daily cytopathology practice.

  14. Alternating Hemiplegia of Childhood mutations have a differential effect on Na(+),K(+)-ATPase activity and ouabain binding.

    PubMed

    Weigand, Karl M; Messchaert, Muriël; Swarts, Herman G P; Russel, Frans G M; Koenderink, Jan B

    2014-07-01

    De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na(+),K(+)-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na(+),K(+)-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K(+)/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na(+),K(+)-ATPase. Functional impairment of Na(+),K(+)-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.

  15. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor

    PubMed Central

    Amsalem, Ayelet R.; Marom, Barak; Shapira, Keren E.; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I.; Ehrlich, Marcelo

    2016-01-01

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and –SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3’ terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  16. [Differential diagnosis of reduced uptake images revealed by bone scan: about a case of acute lymphoblastic leukemia].

    PubMed

    Bahadi, Nisrine; Biyi, Abdelhamid; Oueriagli, Salah Nabih; Doudouh, Abderrahim

    2016-01-01

    If increased uptake during bone scan usually bring to light many bone pathologies, reduced uptakes are a rare occurrence and they require careful analysis to avoid erroneous interpretations. We report the case of a 17-year old admitted with diffuse bone pain, hypercalcemia and thrombopenia. Bone scan showed areas of low uptakes. Bone marrow tests allowed the diagnosis of acute lymphoblastic leukemia. This case report aims to discuss the main differential diagnoses based on such bone scan abnormalities.

  17. Gender-Differentiated Parenting Revisited: Meta-Analysis Reveals Very Few Differences in Parental Control of Boys and Girls.

    PubMed

    Endendijk, Joyce J; Groeneveld, Marleen G; Bakermans-Kranenburg, Marian J; Mesman, Judi

    2016-01-01

    Although various theories describe mechanisms leading to differential parenting of boys and girls, there is no consensus about the extent to which parents do treat their sons and daughters differently. The last meta-analyses on the subject were conducted more than fifteen years ago, and changes in gender-specific child rearing in the past decade are quite plausible. In the current set of meta-analyses, based on 126 observational studies (15,034 families), we examined mothers' and fathers' differential use of autonomy-supportive and controlling strategies with boys and girls, and the role of moderators related to the decade in which the study was conducted, the observational context, and sample characteristics. Databases of Web of Science, ERIC, PsychInfo, Online Contents, Picarta, and Proquest were searched for studies examining differences in observed parental control of boys and girls between the ages of 0 and 18 years. Few differences were found in parents' use of control with boys and girls. Parents were slightly more controlling with boys than with girls, but the effect size was negligible (d = 0.08). The effect was larger, but still small, in normative groups and in samples with younger children. No overall effect for gender-differentiated autonomy-supportive strategies was found (d = 0.03). A significant effect of time emerged: studies published in the 1970s and 1980s reported more autonomy-supportive strategies with boys than toward girls, but from 1990 onwards parents showed somewhat more autonomy-supportive strategies with girls than toward boys. Taking into account parents' gender stereotypes might uncover subgroups of families where gender-differentiated control is salient, but based on our systematic review of the currently available large data base we conclude that in general the differences between parenting of boys versus girls are minimal.

  18. Gender-Differentiated Parenting Revisited: Meta-Analysis Reveals Very Few Differences in Parental Control of Boys and Girls

    PubMed Central

    Endendijk, Joyce J.; Groeneveld, Marleen G.; Bakermans-Kranenburg, Marian J.; Mesman, Judi

    2016-01-01

    Although various theories describe mechanisms leading to differential parenting of boys and girls, there is no consensus about the extent to which parents do treat their sons and daughters differently. The last meta-analyses on the subject were conducted more than fifteen years ago, and changes in gender-specific child rearing in the past decade are quite plausible. In the current set of meta-analyses, based on 126 observational studies (15,034 families), we examined mothers’ and fathers’ differential use of autonomy-supportive and controlling strategies with boys and girls, and the role of moderators related to the decade in which the study was conducted, the observational context, and sample characteristics. Databases of Web of Science, ERIC, PsychInfo, Online Contents, Picarta, and Proquest were searched for studies examining differences in observed parental control of boys and girls between the ages of 0 and 18 years. Few differences were found in parents’ use of control with boys and girls. Parents were slightly more controlling with boys than with girls, but the effect size was negligible (d = 0.08). The effect was larger, but still small, in normative groups and in samples with younger children. No overall effect for gender-differentiated autonomy-supportive strategies was found (d = 0.03). A significant effect of time emerged: studies published in the 1970s and 1980s reported more autonomy-supportive strategies with boys than toward girls, but from 1990 onwards parents showed somewhat more autonomy-supportive strategies with girls than toward boys. Taking into account parents’ gender stereotypes might uncover subgroups of families where gender-differentiated control is salient, but based on our systematic review of the currently available large data base we conclude that in general the differences between parenting of boys versus girls are minimal. PMID:27416099

  19. Longitudinal Transcriptome Analysis Reveals a Sustained Differential Gene Expression Signature in Patients Treated for Acute Lyme Disease

    PubMed Central

    Bouquet, Jerome; Soloski, Mark J.; Swei, Andrea; Cheadle, Chris; Federman, Scot; Billaud, Jean-Noel; Rebman, Alison W.; Kabre, Beniwende; Halpert, Richard; Boorgula, Meher

    2016-01-01

    ABSTRACT Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi, and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the “window period” of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets. PMID:26873097

  20. Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis (2-D DIGE).

    PubMed

    Gao, Liyan; Wang, Aili; Li, Xiaohui; Dong, Kun; Wang, Ke; Appels, Rudi; Ma, Wujun; Yan, Yueming

    2009-12-01

    Comparative proteomics analysis offers a new approach to identify differential proteins among different wheat genotypes and developmental stages. In this study, the non-prolamin expression profiles during grain development of two common or bread wheat cultivars (Triticum aestivum L.), Jing 411 and Sunstate, with different quality properties were analyzed using two-dimensional difference gel electrophoresis (2-D DIGE). Five grain developmental stages during the post-anthesis period were sampled corresponding to the cumulative averages of daily temperatures ( degrees C: 156 degrees C, 250 degrees C, 354 degrees C, 447 degrees C and 749.5 degrees C). More than 400 differential protein spots detected at one or more of the developmental stages of the two cultivars were monitored, among which 230 proteins were identified by MS. Of the identified proteins, more than 85% were enzymes possessing different physiological functions. A total of 36 differential proteins were characterized between the two varieties, which are likely to be related to wheat quality attributes. About one quarter of the proteins identified expressed in multiple spots with different pIs and molecular masses, implying certain post-translational modifications (PTMs) of proteins such as phosphorylations and glycosylations. The results provide new insights into biochemical mechanisms for grain development and quality.

  1. Comparative Genomic Analysis of Transgenic Poplar Dwarf Mutant Reveals Numerous Differentially Expressed Genes Involved in Energy Flow

    PubMed Central

    Chen, Su; Bai, Shuang; Liu, Guifeng; Li, Huiyu; Jiang, Jing

    2014-01-01

    In our previous research, the Tamarix androssowii LEA gene (Tamarix androssowii late embryogenesis abundant protein Mrna, GenBank ID: DQ663481) was transferred into Populus simonii × Populus nigra. Among the eleven transgenic lines, one exhibited a dwarf phenotype compared to the wild type and other transgenic lines, named dwf1. To uncover the mechanisms underlying this phenotype, digital gene expression libraries were produced from dwf1, wild-type, and other normal transgenic lines, XL-5 and XL-6. Gene expression profile analysis indicated that dwf1 had a unique gene expression pattern in comparison to the other two transgenic lines. Finally, a total of 1246 dwf1-unique differentially expressed genes were identified. These genes were further subjected to gene ontology and pathway analysis. Results indicated that photosynthesis and carbohydrate metabolism related genes were significantly affected. In addition, many transcription factors genes were also differentially expressed in dwf1. These various differentially expressed genes may be critical for dwarf mutant formation; thus, the findings presented here might provide insight for our understanding of the mechanisms of tree growth and development. PMID:25192286

  2. Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases

    PubMed Central

    Gstir, Ronald; Schafferer, Simon; Scheideler, Marcel; Misslinger, Matthias; Griehl, Matthias; Daschil, Nina; Humpel, Christian; Obermair, Gerald J.; Schmuckermair, Claudia; Striessnig, Joerg; Flucher, Bernhard E.

    2014-01-01

    We have generated a novel, neuro-specific ncRNA microarray, covering 1472 ncRNA species, to investigate their expression in different mouse models for central nervous system diseases. Thereby, we analyzed ncRNA expression in two mouse models with impaired calcium channel activity, implicated in Epilepsy or Parkinson's disease, respectively, as well as in a mouse model mimicking pathophysiological aspects of Alzheimer's disease. We identified well over a hundred differentially expressed ncRNAs, either from known classes of ncRNAs, such as miRNAs or snoRNAs or which represented entirely novel ncRNA species. Several differentially expressed ncRNAs in the calcium channel mouse models were assigned as miRNAs and target genes involved in calcium signaling, thus suggesting feedback regulation of miRNAs by calcium signaling. In the Alzheimer mouse model, we identified two snoRNAs, whose expression was deregulated prior to amyloid plaque formation. Interestingly, the presence of snoRNAs could be detected in cerebral spine fluid samples in humans, thus potentially serving as early diagnostic markers for Alzheimer's disease. In addition to known ncRNAs species, we also identified 63 differentially expressed, entirely novel ncRNA candidates, located in intronic or intergenic regions of the mouse genome, genomic locations, which previously have been shown to harbor the majority of functional ncRNAs. PMID:25344396

  3. Ablation of beta1 integrin in mammary epithelium reveals a key role for integrin in glandular morphogenesis and differentiation.

    PubMed

    Naylor, Matthew J; Li, Na; Cheung, Julia; Lowe, Emma T; Lambert, Elise; Marlow, Rebecca; Wang, Pengbo; Schatzmann, Franziska; Wintermantel, Timothy; Schüetz, Günther; Clarke, Alan R; Mueller, Ulrich; Hynes, Nancy E; Streuli, Charles H

    2005-11-21

    Integrin-mediated adhesion regulates the development and function of a range of tissues; however, little is known about its role in glandular epithelium. To assess the contribution of beta1 integrin, we conditionally deleted its gene in luminal epithelia during different stages of mouse mammary gland development and in cultured primary mammary epithelia. Loss of beta1 integrin in vivo resulted in impaired alveologenesis and lactation. Cultured beta1 integrin-null cells displayed abnormal focal adhesion function and signal transduction and could not form or maintain polarized acini. In vivo, epithelial cells became detached from the extracellular matrix but remained associated with each other and did not undergo overt apoptosis. beta1 integrin-null mammary epithelial cells did not differentiate in response to prolactin stimulation because of defective Stat5 activation. In mice where beta1 integrin was deleted after the initiation of differentiation, fewer defects in alveolar morphology occurred, yet major deficiencies were also observed in milk protein and milk fat production and Stat5 activation, indicating a permissive role for beta1 integrins in prolactin signaling. This study demonstrates that beta1 integrin is critical for the alveolar morphogenesis of a glandular epithelium and for maintenance of its differentiated function. Moreover, it provides genetic evidence for the cooperation between integrin and cytokine signaling pathways.

  4. TRA-1 ChIP-seq reveals regulators of sexual differentiation and multilevel feedback in nematode sex determination

    PubMed Central

    Berkseth, Matt; Ikegami, Kohta; Arur, Swathi; Lieb, Jason D.; Zarkower, David

    2013-01-01

    How sexual regulators translate global sexual fate into appropriate local sexual differentiation events is perhaps the least understood aspect of sexual development. Here we have used ChIP followed by deep sequencing (ChIP-seq) to identify direct targets of the nematode global sexual regulator Transformer 1 (TRA-1), a transcription factor acting at the interface between organism-wide and cell-specific sexual regulation to control all sex-specific somatic differentiation events. We identified 184 TRA-1–binding sites in Caenorhabditis elegans, many with temporal- and/or tissue-specific TRA-1 association. We also identified 78 TRA-1–binding sites in the related nematode Caenorhabditis briggsae, 19 of which are conserved between the two species. Some DNA segments containing TRA-1–binding sites drive male-specific expression patterns, and RNAi depletion of some genes adjacent to TRA-1–binding sites results in defects in male sexual development. TRA-1 binds to sites adjacent to a number of heterochronic regulatory genes, some of which drive male-specific expression, suggesting that TRA-1 imposes sex specificity on developmental timing. We also found evidence for TRA-1 feedback regulation of the global sex-determination pathway: TRA-1 binds its own locus and those of multiple upstream masculinizing genes, and most of these associations are conserved in C. briggsae. Thus, TRA-1 coordinates sexual development by reinforcing the sex-determination decision and directing downstream sexual differentiation events. PMID:24046365

  5. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells.

    PubMed

    Kowalczyk, Monika S; Tirosh, Itay; Heckl, Dirk; Rao, Tata Nageswara; Dixit, Atray; Haas, Brian J; Schneider, Rebekka K; Wagers, Amy J; Ebert, Benjamin L; Regev, Aviv

    2015-12-01

    Both intrinsic cell state changes and variations in the composition of stem cell populations have been implicated as contributors to aging. We used single-cell RNA-seq to dissect variability in hematopoietic stem cell (HSC) and hematopoietic progenitor cell populations from young and old mice from two strains. We found that cell cycle dominates the variability within each population and that there is a lower frequency of cells in the G1 phase among old compared with young long-term HSCs, suggesting that they traverse through G1 faster. Moreover, transcriptional changes in HSCs during aging are inversely related to those upon HSC differentiation, such that old short-term (ST) HSCs resemble young long-term (LT-HSCs), suggesting that they exist in a less differentiated state. Our results indicate both compositional changes and intrinsic, population-wide changes with age and are consistent with a model where a relationship between cell cycle progression and self-renewal versus differentiation of HSCs is affected by aging and may contribute to the functional decline of old HSCs.

  6. Differential gene expression for Curvularia eragrostidis pathogenic incidence in crabgrass (Digitaria sanguinalis) revealed by cDNA-AFLP analysis.

    PubMed

    Wang, Jianshu; Wang, Xuemin; Yuan, Bohua; Qiang, Sheng

    2013-01-01

    Gene expression profiles of Digitaria sanguinalis infected by Curvularia eragrostidis strain QZ-2000 at two concentrations of conidia and two dew durations were analyzed by cDNA amplified fragment length polymorphisms (cDNA-AFLP). Inoculum strength was more determinant of gene expression than dew duration. A total of 256 primer combinations were used for selective amplification and 1214 transcript-derived fragments (TDFs) were selected for their differential expression. Of these, 518 up-regulated differentially expressed TDFs were identified. Forty-six differential cDNA fragments were chosen to be cloned and 35 of them were successfully cloned and sequenced, of which 25 were homologous to genes of known function according to the GenBank database. Only 6 genes were up-regulated in Curvularia eragrostidis-inoculated D. sanguinalis, with functions involved in signal transduction, energy metabolism, cell growth and development, stress responses, abscisic acid biosynthesis and response. It appears that a few pathways may be important parts of the pathogenic strategy of C. eragrostidis strain QZ-2000 on D. sanguinalis. Our study provides the fundamentals to further study the pathogenic mechanism, screen for optimal C. eragrostidis strains as potential mycoherbicide and apply this product to control D. sanguinalis.

  7. Genetic population differentiation of the blue swimming crab Portunus pelagicus (Portunidae) in Thai waters revealed by RAPD analysis.

    PubMed

    Klinbunga, S; Yuvanatemiya, V; Wongphayak, S; Khetpu, K; Menasveta, P; Khamnamtong, B

    2010-08-17

    Genetic diversity and population differentiation of the blue swimming crab, Portunus pelagicus, in Thailand were analyzed by RAPD analysis. One hundred and twelve RAPD fragments were generated from 109 individuals of P. pelagicus using OPA02, OPA14, OPB10, UBC122, and UBC158 primers. The percentage of polymorphic bands in each geographic sample and that of each primer across overall samples were 72.7-85.0 and 92.0-100%, respectively. Large numbers of polymorphic bands found in the RAPD analysis suggested high genetic diversity of Thai P. pelagicus. The mean genetic distance between samples across all primers was 0.0929-0.2471. Significant geographic heterogeneity was observed across samples overall and between all pairs of geographic samples (P < 0.01 for theta and P < 0.0001 for the exact test), indicating strong genetic differentiation of P. pelagicus in Thai waters, despite its high potential of dispersal. Limited gene flow levels (0.44-1.19 individuals per generation) of Thai P. pelagicus were also observed. A fine scale level of differentiation suggested that P. pelagicus from each geographic sample in Thai waters should be regarded as a separate genetic population and treated as a different exploited stock.

  8. The Hansenula polymorpha MOX gene presents two alternative transcription start points differentially utilized and sensitive to respiratory activity.

    PubMed

    Genu, Victor; Gödecke, Stefanie; Hollenberg, Cornelis P; Pereira, Gonçalo G

    2003-06-01

    The peroxisomal methanol metabolism of Hansenula polymorpha depends on a group of genes that are coordinately regulated. Methanol oxidase (Mox) plays a key role in this pathway and its synthesis has been shown to be regulated at the transcriptional level. MOX expression is strongly repressed on glucose and activated on glycerol or methanol. In this study we have identified two MOX transcripts that are differentially expressed along MOX derepression. The first one, named l-MOX (for longer MOX), starts at position -425, is only weakly and transiently transcribed and is not translated into the Mox protein. The other is the true MOX mRNA, which initiates around position -25. Using a strain bearing multiple copies of MOX(Q1N) and a reporter gene fused to the MOX promoter, regulation of the two transcripts was investigated. Initiation of the true MOX correlates with repression of l-MOX and conditions that are repressive for MOX transcription, such as the inhibition of mitochondrial activity, lead to higher levels of l-MOX expression. This effect was first observed in a mox mutant (Q1N-M8) unable to grow on nonfermentable carbon sources. No function was detected for l-MOX, but its regulation follows a pattern similar to that of catalase, which is essential for methanol metabolism. This suggests that, l-MOX, although precisely regulated, seems to be a remnant of the evolution of the methanol metabolism network.

  9. Global transcription analysis of Krebs tricarboxylic acid cycle mutants reveals an alternating pattern of gene expression and effects on hypoxic and oxidative genes.

    PubMed

    McCammon, Mark T; Epstein, Charles B; Przybyla-Zawislak, Beata; McAlister-Henn, Lee; Butow, Ronald A

    2003-03-01

    To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction. Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism. Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in alpha-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants. This pattern correlated with previously defined TCA cycle growth-enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function. Expression of hypoxic/anaerobic genes was elevated in alpha-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA. These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle.

  10. Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.).

    PubMed

    Wang, Shuzhen; Chen, Wenyue; Xiao, Wenfei; Yang, Changdeng; Xin, Ya; Qiu, Jieren; Hu, Weimin; Ying, Wu; Fu, Yaping; Tong, Jianxin; Hu, Guocheng; Chen, Zhongzhong; Fang, Xianping; Yu, Hong; Lai, Wenguo; Ruan, Songlin; Ma, Huasheng

    2015-01-01

    Rice hull, the outer cover of the rice grain, determines grain shape and size. Changes in the rice hull proteome in different growth stages may reflect the underlying mechanisms involved in grain development. To better understand these changes, isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS was used to detect statistically significant changes in the rice hull proteome in the booting, flowering, and milk-ripe growth stages. Differentially expressed proteins were analyzed to predict their potential functions during development. Gene ontology (GO) terms and pathways were used to evaluate the biological mechanisms involved in rice hull at the three growth stages. In total, 5,268 proteins were detected and characterized, of which 563 were differentially expressed across the development stages. The results showed that the flowering and milk-ripe stage proteomes were more similar to each other (r=0.61) than either was to the booting stage proteome. A GO enrichment analysis of the differentially expressed proteins was used to predict their roles during rice hull development. The potential functions of 25 significantly differentially expressed proteins were used to evaluate their possible roles at various growth stages. Among these proteins, an unannotated protein (Q7X8A1) was found to be overexpressed especially in the flowering stage, while a putative uncharacterized protein (B8BF94) and an aldehyde dehydrogenase (Q9FPK6) were overexpressed only in the milk-ripe stage. Pathways regulated by differentially expressed proteins were also analyzed. Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), and two magnesium-chelatase subunits, ChlD (Q6ATS0), and ChlI (Q53RM0), were associated with chlorophyll biosynthesis at different developmental stages. The expression of Q9SDJ2 in the flowering and milk-ripe stages was validated by qRT-PCR. The 25 candidate proteins may be pivotal markers for controlling rice hull development at various

  11. Moderate Genetic Diversity and Genetic Differentiation in the Relict Tree Liquidambar formosana Hance Revealed by Genic Simple Sequence Repeat Markers

    PubMed Central

    Sun, Rongxi; Lin, Furong; Huang, Ping; Zheng, Yongqi

    2016-01-01

    Chinese sweetgum (Liquidambar formosana) is a relatively fast-growing ecological pioneer species. It is widely used for multiple purposes. To assess the genetic diversity and genetic differentiation of the species, genic SSR markers were mined from transcriptome data for subsequent analysis of the genetic diversity and population structure of natural populations. A total of 10645 potential genic SSR loci were identified in 80482 unigenes. The average frequency was one SSR per 5.12 kb, and the dinucleotide unit was the most abundant motif. A total of 67 alleles were found, with a mean of 6.091 alleles per locus and a mean polymorphism information content of 0.390. Moreover, the species exhibited a relatively moderate level of genetic diversity (He = 0.399), with the highest was found in population XY (He = 0.469). At the regional level, the southwestern region displayed the highest genetic diversity (He = 0.435) and the largest number of private alleles (n = 5), which indicated that the Southwestern region may be the diversity hot spot of L. formosana. The AMOVA results showed that variation within populations (94.02%) was significantly higher than among populations (5.98%), which was in agreement with the coefficient of genetic differentiation (Fst = 0.076). According to the UPGMA analysis and principal coordinate analysis and confirmed by the assignment test, 25 populations could be divided into three groups, and there were different degrees of introgression among populations. No correlation was found between genetic distance and geographic distance (P > 0.05). These results provided further evidence that geographic isolation was not the primary factor leading to the moderate genetic differentiation of L. formosana. As most of the genetic diversity of L. formosana exists among individuals within a population, individual plant selection would be an effective way to use natural variation in genetic improvement programs. This would be helpful to not only protect the

  12. Single Cell Analysis Reveals Concomitant Transcription of Pluripotent and Lineage Markers During the Early Steps of Differentiation of Embryonic Stem Cells.

    PubMed

    Lanctôt, Christian

    2015-10-01

    The differentiation of embryonic stem cells is associated with extensive changes in gene expression. It is not yet clear whether these changes are the result of binary switch-like mechanisms or that of continuous and progressive variation. Here, I have used immunostaining and single molecule RNA fluorescence in situ hybridization (FISH) to assess changes in the expression of the well-known pluripotency-associated gene Pou5f1 (also known as Oct4) and early differentiation markers Sox1 and T-brachyury in single cells during the early steps of differentiation of mouse embryonic stem cells. I found extensive overlap between the expression of Pou5f1/Sox1 or Pou5f1/T-brachyury shortly after the initiation of differentiation towards either the neuronal or the mesendodermal lineage, but no evidence of correlation between their respective expression levels. Quantitative analysis of transcriptional output at the sites of nascent transcription revealed that Pou5f1 and Sox1 were transcribed in pulses and that embryonic stem cell differentiation was accompanied by changes in pulsing frequencies. The progressive induction of Sox1 was further associated with an increase in the average size of individual transcriptional bursts. Surprisingly, single cells that actively and simultaneously transcribe both the pluripotency- and the lineage-associated genes could easily be found in the differentiating population. The results presented here show for the first time that lineage priming can occur in cells that are actively transcribing a pluripotent marker. Furthermore, they suggest that this process is associated with changes in transcriptional dynamics.

  13. Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay

    NASA Astrophysics Data System (ADS)

    Zhou, Yitian; Zhou, Ping; Xin, Yinqiang; Wang, Jie; Zhu, Zhiqiang; Hu, Ji; Wei, Shicheng; Ma, Hongwei

    2014-11-01

    Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

  14. Disturbed local auxin homeostasis enhances cellular anisotropy and reveals alternative wiring of auxin-ethylene crosstalk in Brachypodium distachyon seminal roots.

    PubMed

    Pacheco-Villalobos, David; Sankar, Martial; Ljung, Karin; Hardtke, Christian S

    2013-06-01

    cell elongation, as suggested by our observations. Thus, our results reveal a delicate homeostasis of local auxin and ethylene activity to control cell elongation in Brachypodium roots and suggest alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis.

  15. The Polymyxin Ceftazidime Oxford Medium as an alternative selective and differential medium for isolation of Listeria monocytogenes from raw or unpasteurized food.

    PubMed

    Martínez-Gonzáles, N E; Martínez-Chávez, L; Martínez-Cárdenas, C; Cabrera-Díaz, E; Castillo, A

    2014-04-01

    The Polymyxin Ceftazidime Oxford Medium (PCOM) was developed to recover Listeria monocytogenes from raw or unpasteurized foods. It contains esculin-ferric ammonium citrate as indicator system for Listeria growth, and ceftazidime and polymyxin B as selective agents, which are available in several Latin American countries. Comparison of PCOM, Modified Oxford Medium (MOX) and Tryptic Soy agar with 0.6% yeast extract (TSAYE) indicated that both selective media were equally effective at recovering four individual strains of L. monocytogenes (Scott A, V7, California and broccoli), and a mixture of these strains (LMM) (P > 0.05). The ability of PCOM, MOX, TSAYE and TSAYE supplemented with 4% NaCl to recover heat, acid and freeze-damaged LMM was similar for all media (P > 0.05). The PCOM proved to be effective at isolating colonies of LMM from inoculated raw beef chunks, unpasteurized orange juice, cabbage, and Mexican-style cheese by direct plating and by the US Department of Agriculture's Food Safety and Inspection Service enrichment method. Differentiation of L. monocytogenes colonies was easier on PCOM than on MOX for foods with high levels of background microbiota. Based on the evaluations performed on foods naturally contaminated with L. monocytogenes, PCOM was a more economical alternative than MOX for selective and differential isolation of Listeria from raw or unpasteurized foods.

  16. Differential proteomic analysis of midguts from Nosema ceranae-infected honeybees reveals manipulation of key host functions.

    PubMed

    Vidau, Cyril; Panek, Johan; Texier, Catherine; Biron, David G; Belzunces, Luc P; Le Gall, Morgane; Broussard, Cédric; Delbac, Frédéric; El Alaoui, Hicham

    2014-09-01

    Many invasive pathogens effectively bypass the insect defenses to ensure the completion of their life cycle. Among those, an invasive microsporidian species, Nosema ceranae, can cause nosemosis in honeybees. N. ceranae was first described in the Asian honeybee Apis cerana and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the first barrier against N. ceranae attacks. To bring proteomics data on honeybee/N. ceranae crosstalk and more precisely to decipher the worker honeybee midgut response after an oral inoculation of N. ceranae (10days post-infection), we used 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) combined with mass spectrometry. Forty-five protein spots produced by the infected worker honeybee group were shown to be differentially expressed when compared to the uninfected group; 14 were subsequently identified by mass spectrometry. N. ceranae mainly caused a modulation of proteins involved in three key host biological functions: (i) energy production, (ii) innate immunity (reactive oxygen stress) and (iii) protein regulation. The modulation of these host biological functions suggests that N. ceranae creates a zone of "metabolic habitat modification" in the honeybee midgut favoring its development by enhancing availability of nutrients and reducing the worker honeybee defense.

  17. Proteomic analysis of human brain microvascular endothelial cells reveals differential protein expression in response to enterovirus 71 infection.

    PubMed

    Luo, Wenying; Zhong, Jiayu; Zhao, Wei; Liu, Jianjun; Zhang, Renli; Peng, Liang; Hong, Wenxu; Huang, Sheng He; Cao, Hong

    2015-01-01

    2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC), to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS) analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications.

  18. Transcriptome analysis in Coffea eugenioides, an Arabica coffee ancestor, reveals differentially expressed genes in leaves and fruits.

    PubMed

    Yuyama, Priscila Mary; Reis Júnior, Osvaldo; Ivamoto, Suzana Tiemi; Domingues, Douglas Silva; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães; Charmetant, Pierre; Leroy, Thierry; Pereira, Luiz Filipe Protasio

    2016-02-01

    Studies in diploid parental species of polyploid plants are important to understand their contributions to the formation of plant and species evolution. Coffea eugenioides is a diploid species that is considered to be an ancestor of allopolyploid Coffea arabica together with Coffea canephora. Despite its importance in the evolutionary history of the main economic species of coffee, no study has focused on C. eugenioides molecular genetics. RNA-seq creates the possibility to generate reference transcriptomes and identify coding genes and potential candidates related to important agronomic traits. Therefore, the main objectives were to obtain a global overview of transcriptionally active genes in this species using next-generation sequencing and to analyze specific genes that were highly expressed in leaves and fruits with potential exploratory characteristics for breeding and understanding the evolutionary biology of coffee. A de novo assembly generated 36,935 contigs that were annotated using eight databases. We observed a total of ~5000 differentially expressed genes between leaves and fruits. Several genes exclusively expressed in fruits did not exhibit similarities with sequences in any database. We selected ten differentially expressed unigenes in leaves and fruits to evaluate transcriptional profiles using qPCR. Our study provides the first gene catalog for C. eugenioides and enhances the knowledge concerning the mechanisms involved in the C. arabica homeologous. Furthermore, this work will open new avenues for studies into specific genes and pathways in this species, especially related to fruit, and our data have potential value in assisted breeding applications.

  19. Differential display RT-PCR reveals genes associated with lithium-induced neuritogenesis in SK-N-MC cells.

    PubMed

    Italia, Jennifer; Mukhopadhyaya, Rita; Rajadhyaksha, Medha S

    2011-10-01

    Lithium is shown to be neurotrophic and protective against variety of environmental stresses both in vitro as well as in vivo. In view of the wider clinical applications, it is necessary to examine alterations in levels of expression of genes affected by lithium. Lithium induces neuritogenesis in human neuroblastoma cell line SK-N-MC. Our aim was to elucidate genes involved in lithium-induced neuritogenesis using SK-N-MC cells. The differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) technique was used to study gene expression profiles in SK-N-MC cells undergoing lithium-induced neuritogenesis. Differential expression of genes in control and lithium (2.5 mM, 24 h)-treated cells was compared by display of cDNAs generated by reverse transcription of mRNA followed by PCR using arbitrary primers. Expression of four genes was altered in lithium-treated cells. Real-time PCR was done to confirm the levels of expression of each of these genes using specific primers. Lithium significantly up-regulated NCAM, a molecule known to stimulate neuritogenesis, occludin, a molecule participating in tight junctions and PKD2, a molecule known to modulate calcium transport. ANP 32c, a gene whose function is not fully known yet, was found to be down-regulated by lithium. This is the first report demonstrating altered levels of expression of these genes in lithium-induced neuritogenesis and contributes four hitherto unreported candidates possibly involved in the process.

  20. Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host.

    PubMed

    Mikolajczak, Sebastian A; Silva-Rivera, Hilda; Peng, Xinxia; Tarun, Alice S; Camargo, Nelly; Jacobs-Lorena, Vanessa; Daly, Thomas M; Bergman, Lawrence W; de la Vega, Patricia; Williams, Jack; Aly, Ahmed S I; Kappe, Stefan H I

    2008-10-01

    The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes).

  1. Efficient induction of dopaminergic neuron differentiation from induced pluripotent stem cells reveals impaired mitophagy in PARK2 neurons.

    PubMed

    Suzuki, Sadafumi; Akamatsu, Wado; Kisa, Fumihiko; Sone, Takefumi; Ishikawa, Kei-Ichi; Kuzumaki, Naoko; Katayama, Hiroyuki; Miyawaki, Atsushi; Hattori, Nobutaka; Okano, Hideyuki

    2017-01-29

    Patient-specific induced pluripotent stem cells (iPSCs) show promise for use as tools for in vitro modeling of Parkinson's disease. We sought to improve the efficiency of dopaminergic (DA) neuron induction from iPSCs by the using surface markers expressed in DA progenitors to increase the significance of the phenotypic analysis. By sorting for a CD184(high)/CD44(-) fraction during neural differentiation, we obtained a population of cells that were enriched in DA neuron precursor cells and achieved higher differentiation efficiencies than those obtained through the same protocol without sorting. This high efficiency method of DA neuronal induction enabled reliable detection of reactive oxygen species (ROS) accumulation and vulnerable phenotypes in PARK2 iPSCs-derived DA neurons. We additionally established a quantitative system using the mt-mKeima reporter system to monitor mitophagy in which mitochondria fuse with lysosomes and, by combining this system with the method of DA neuronal induction described above, determined that mitophagy is impaired in PARK2 neurons. These findings suggest that the efficiency of DA neuron induction is important for the precise detection of cellular phenotypes in modeling Parkinson's disease.

  2. Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

    PubMed

    Gómez-Molero, Emilia; de Boer, Albert D; Dekker, Henk L; Moreno-Martínez, Ana; Kraneveld, Eef A; Ichsan; Chauhan, Neeraj; Weig, Michael; de Soet, Johannes J; de Koster, Chris G; Bader, Oliver; de Groot, Piet W J

    2015-12-01

    Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.

  3. Differential effects of Zn exposure on male and female oysters (Crassostrea angulata) as revealed by label-free quantitative proteomics.

    PubMed

    Luo, Lianzhong; Zhang, Qinghong; Kong, Xue; Huang, Heqing; You, Weiwei; Ke, Caihuan

    2017-03-17

    Oysters accumulate Zn as an adaptation to Zn exposure; however, it is not known whether male and female oysters respond differently to Zn exposure. Here, proteomic and real-time polymerase chain reaction analyses were used to investigate differential responses of male and female oysters (Crassostrea angulata) to Zn exposure. After exposure to 50 µg.L(-1) or 500 µg.L(-1) Zn for 30 days, gonads of female oysters accumulated more Zn than those of males, and gonadal development was accelerated in females but was abnormal in males. Differentially expressed proteins after exposure to Zn were identified and shown to function in Zn transport, Ca transport, phosphate metabolism, energy metabolism, immune regulation, oxidative stress responses, gene expression regulation, and fat metabolism. Proteins with functions in Zn transportation and storage, and multi-functional proteins, such as hemicentin-1 and histidinol dehydrogenase, were expressed at significantly higher levels in the gonads of female than male oysters after Zn exposure. This article is protected by copyright. All rights reserved.

  4. Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation.

    PubMed

    De Cegli, Rossella; Iacobacci, Simona; Flore, Gemma; Gambardella, Gennaro; Mao, Lei; Cutillo, Luisa; Lauria, Mario; Klose, Joachim; Illingworth, Elizabeth; Banfi, Sandro; di Bernardo, Diego

    2013-01-01

    Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology 'reverse engineering' approaches. We 'reverse engineered' an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression ('hubs'). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central 'hub' of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation.

  5. Human 45,X Fibroblast Transcriptome Reveals Distinct Differentially Expressed Genes Including Long Noncoding RNAs Potentially Associated with the Pathophysiology of Turner Syndrome

    PubMed Central

    Patowary, Ashok; Scaria, Vinod; Sivasubbu, Sridhar; Deobagkar, Deepti D.

    2014-01-01

    Turner syndrome is a chromosomal abnormality characterized by the absence of whole or part of the X chromosome in females. This X aneuploidy condition is associated with a diverse set of clinical phenotypes such as gonadal dysfunction, short stature, osteoporosis and Type II diabetes mellitus, among others. These phenotypes differ in their severity and penetrance among the affected individuals. Haploinsufficiency for a few X linked genes has been associated with some of these disease phenotypes. RNA sequencing can provide valuable insights to understand molecular mechanism of disease process. In the current study, we have analysed the transcriptome profiles of human untransformed 45,X and 46,XX fibroblast cells and identified differential expression of genes in these two karyotypes. Functional analysis revealed that these differentially expressing genes are associated with bone differentiation, glucose metabolism and gonadal development pathways. We also report differential expression of lincRNAs in X monosomic cells. Our observations provide a basis for evaluation of cellular and molecular mechanism(s) in the establishment of Turner syndrome phenotypes. PMID:24932682

  6. Human 45,X fibroblast transcriptome reveals distinct differentially expressed genes including long noncoding RNAs potentially associated with the pathophysiology of Turner syndrome.

    PubMed

    Rajpathak, Shriram N; Vellarikkal, Shamsudheen Karuthedath; Patowary, Ashok; Scaria, Vinod; Sivasubbu, Sridhar; Deobagkar, Deepti D

    2014-01-01

    Turner syndrome is a chromosomal abnormality characterized by the absence of whole or part of the X chromosome in females. This X aneuploidy condition is associated with a diverse set of clinical phenotypes such as gonadal dysfunction, short stature, osteoporosis and Type II diabetes mellitus, among others. These phenotypes differ in their severity and penetrance among the affected individuals. Haploinsufficiency for a few X linked genes has been associated with some of these disease phenotypes. RNA sequencing can provide valuable insights to understand molecular mechanism of disease process. In the current study, we have analysed the transcriptome profiles of human untransformed 45,X and 46,XX fibroblast cells and identified differential expression of genes in these two karyotypes. Functional analysis revealed that these differentially expressing genes are associated with bone differentiation, glucose metabolism and gonadal development pathways. We also report differential expression of lincRNAs in X monosomic cells. Our observations provide a basis for evaluation of cellular and molecular mechanism(s) in the establishment of Turner syndrome phenotypes.

  7. Simultaneous steady-state and dynamic 13C NMR can differentiate alternative routes of pyruvate metabolism in living cancer cells.

    PubMed

    Yang, Chendong; Harrison, Crystal; Jin, Eunsook S; Chuang, David T; Sherry, A Dean; Malloy, Craig R; Merritt, Matthew E; DeBerardinis, Ralph J

    2014-02-28

    Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of (13)C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized (13)C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined (13)C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-(13)C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-(13)C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[(13)C]O3(-) and [1-(13)C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-(13)C]pyruvate. Quantitation of hyperpolarized H[(13)C]O3(-) provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[(13)C]O3(-) appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1-(13)C]pyruvate metabolism, enhancing exchanges with [1-(13)C]lactate and suppressing H[(13)C]O3(-) formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining (13)C isotopomer analyses and dynamic hyperpolarized (13)C spectroscopy may enable

  8. A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

    PubMed Central

    Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R.; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

    2016-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders. PMID:27739443

  9. A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells.

    PubMed

    Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

    2016-10-14

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.

  10. Global Mapping of Cell Type–Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

    PubMed Central

    Yu, Jing; Hirose-Yotsuya, Lisa; Take, Kazumi; Sun, Wei; Iwabu, Masato; Okada-Iwabu, Miki; Fujita, Takanori; Aoyama, Tomohisa; Tsutsumi, Shuichi; Ueki, Kohjiro; Kodama, Tatsuhiko; Sakai, Juro; Aburatani, Hiroyuki; Kadowaki, Takashi

    2011-01-01

    Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study

  11. Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternative Conformation of NADPH that may be Linked to Trimethoprim Resistance

    SciTech Connect

    Frey, K.; Liu, J; Lombardo, M; Bolstad, D; Wright, D; Anderson, A

    2009-01-01

    Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.

  12. Proteomic analysis reveals contrasting stress response to uranium in two nitrogen-fixing Anabaena strains, differentially tolerant to uranium.

    PubMed

    Panda, Bandita; Basu, Bhakti; Acharya, Celin; Rajaram, Hema; Apte, Shree Kumar

    2017-01-01

    Two strains of the nitrogen-fixing cyanobacterium Anabaena, native to Indian paddy fields, displayed differential sensitivity to exposure to uranyl carbonate at neutral pH. Anabaena sp. strain PCC 7120 and Anabaena sp. strain L-31 displayed 50% reduction in survival (LD50 dose), following 3h exposure to 75μM and 200μM uranyl carbonate, respectively. Uranium responsive proteome alterations were visualized by 2D gel electrophoresis, followed by protein identification by MALDI-ToF mass spectrometry. The two strains displayed significant differences in levels of proteins associated with photosynthesis, carbon metabolism, and oxidative stress alleviation, commensurate with their uranium tolerance. Higher uranium tolerance of Anabaena sp. strain L-31 could be attributed to sustained photosynthesis and carbon metabolism and superior oxidative stress defense, as compared to the uranium sensitive Anabaena sp. strain PCC 7120.

  13. The Kinematics of Plant Nutation Reveals a Simple Relation between Curvature and the Orientation of Differential Growth

    PubMed Central

    Bastien, Renaud; Meroz, Yasmine

    2016-01-01

    Nutation is an oscillatory movement that plants display during their development. Despite its ubiquity among plants movements, the relation between the observed movement and the underlying biological mechanisms remains unclear. Here we show that the kinematics of the full organ in 3D give a simple picture of plant nutation, where the orientation of the curvature along the main axis of the organ aligns with the direction of maximal differential growth. Within this framework we reexamine the validity of widely used experimental measurements of the apical tip as markers of growth dynamics. We show that though this relation is correct under certain conditions, it does not generally hold, and is not sufficient to uncover the specific role of each mechanism. As an example we re-interpret previously measured experimental observations using our model. PMID:27923062

  14. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

    PubMed

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.

  15. Small RNA Sequencing Reveals Differential miRNA Expression in the Early Development of Broccoli (Brassica oleracea var. italica) Pollen

    PubMed Central

    Li, Hui; Wang, Yu; Wu, Mei; Li, Lihong; Jin, Chuan; Zhang, Qingli; Chen, Chengbin; Song, Wenqin; Wang, Chunguo

    2017-01-01

    Pollen development is an important and complex biological process in the sexual reproduction of flowering plants. Although the cytological characteristics of pollen development are well defined, the regulation of its early stages remains largely unknown. In the present study, miRNAs were explored in the early development of broccoli (Brassica oleracea var. italica) pollen. A total of 333 known miRNAs that originated from 235 miRNA families were detected. Fifty-five novel miRNA candidates were identified. Sixty of the 333 known miRNAs and 49 of the 55 predicted novel miRNAs exhibited significantly differential expression profiling in the three distinct developmental stages of broccoli pollen. Among these differentially expressed miRNAs, miRNAs that would be involved in the developmental phase transition from uninucleate microspores to binucleate pollen grains or from binucleate to trinucleate pollen grains were identified. miRNAs that showed significantly enriched expression in a specific early stage of broccoli pollen development were also observed. In addition, 552 targets for 127 known miRNAs and 69 targets for 40 predicted novel miRNAs were bioinformatically identified. Functional annotation and GO (Gene Ontology) analysis indicated that the putative miRNA targets showed significant enrichment in GO terms that were related to plant organ formation and morphogenesis. Some of enriched GO terms were detected for the targets directly involved in plant male reproduction development. These findings provided new insights into the functions of miRNA-mediated regulatory networks in broccoli pollen development. PMID:28392797

  16. Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle.

    PubMed

    Murphy, Robyn M; Xu, Hongyang; Latchman, Heidy; Larkins, Noni T; Gooley, Paul R; Stapleton, David I

    2012-12-01

    To understand how glycogen affects skeletal muscle physiology, we examined enzymes essential for muscle glycogen synthesis and degradation using single fibers from quiescent and stimulated rat skeletal muscle. Presenting a shift in paradigm, we show these proteins are differentially associated with glycogen granules. Protein diffusibility and/or abundance of glycogenin, glycogen branching enzyme (GBE), debranching enzyme (GDE), phosphorylase (GP), and synthase (GS) were examined in fibers isolated from rat fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscle. GDE and GP proteins were more abundant (~10- to 100-fold) in fibers from EDL compared with SOL muscle. GS and glycogenin proteins were similar between muscles while GBE had an approximately fourfold greater abundance in SOL muscle. Mechanically skinned fibers exposed to physiological buffer for 10 min showed ~70% total pools of GBE and GP were diffusible (nonbound), whereas GDE and GS were considerably less diffusible. Intense in vitro stimulation, sufficient to elicit a ~50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (~15-60%) and decreased GBE diffusibility (~20%). Amylase treatment, which breaks α-1,4 linkages of glycogen, indicated differential diffusibilities and hence glycogen associations of GDE and GS. Membrane solubilization (1% Triton-X-100) allowed a small additional amount of GDE and GS to diffuse from fibers, suggesting the majority of nonglycogen-associated GDE/GS is associated with myofibrillar/contractile network of muscle rather than membranes. Given differences in enzymes required for glycogen metabolism, the current findings suggest glycogen particles have fiber-type-dependent structures. The greater catabolic potential of glycogen breakdown in fast-twitch fibers may account for different contraction induced rates of glycogen utilization.

  17. High-throughput sequencing reveals differential expression of miRNAs in intestine from sea cucumber during aestivation.

    PubMed

    Chen, Muyan; Zhang, Xiumei; Liu, Jianning; Storey, Kenneth B

    2013-01-01

    The regulatory role of miRNA in gene expression is an emerging hot new topic in the control of hypometabolism. Sea cucumber aestivation is a complicated physiological process that includes obvious hypometabolism as evidenced by a decrease in the rates of oxygen consumption and ammonia nitrogen excretion, as well as a serious degeneration of the intestine into a very tiny filament. To determine whether miRNAs play regulatory roles in this process, the present study analyzed profiles of miRNA expression in the intestine of the sea cucumber (Apostichopus japonicus), using Solexa deep sequencing technology. We identified 308 sea cucumber miRNAs, including 18 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA) after at least 15 days of continuous torpor, were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to the active state). We identified 42 differentially expressed miRNAs [RPM (reads per million) >10, |FC| (|fold change|) ≥ 1, FDR (false discovery rate) <0.01] during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-200-3p, miR-2004, miR-2010, miR-22, miR-252a, miR-252a-3p and miR-92 were significantly over-expressed during deep aestivation compared with non-aestivation animals. Preliminary analyses of their putative target genes and GO analysis suggest that these miRNAs could play important roles in global transcriptional depression and cell differentiation during aestivation. High-throughput sequencing data and microarray data have been submitted to GEO database.

  18. High-Throughput Sequencing Reveals Differential Expression of miRNAs in Intestine from Sea Cucumber during Aestivation

    PubMed Central

    Chen, Muyan; Zhang, Xiumei; Liu, Jianning; Storey, Kenneth B.

    2013-01-01

    The regulatory role of miRNA in gene expression is an emerging hot new topic in the control of hypometabolism. Sea cucumber aestivation is a complicated physiological process that includes obvious hypometabolism as evidenced by a decrease in the rates of oxygen consumption and ammonia nitrogen excretion, as well as a serious degeneration of the intestine into a very tiny filament. To determine whether miRNAs play regulatory roles in this process, the present study analyzed profiles of miRNA expression in the intestine of the sea cucumber (Apostichopus japonicus), using Solexa deep sequencing technology. We identified 308 sea cucumber miRNAs, including 18 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA) after at least 15 days of continuous torpor, were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to the active state). We identified 42 differentially expressed miRNAs [RPM (reads per million) >10, |FC| (|fold change|) ≥1, FDR (false discovery rate) <0.01] during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-200-3p, miR-2004, miR-2010, miR-22, miR-252a, miR-252a-3p and miR-92 were significantly over-expressed during deep aestivation compared with non-aestivation animals. Preliminary analyses of their putative target genes and GO analysis suggest that these miRNAs could play important roles in global transcriptional depression and cell differentiation during aestivation. High-throughput sequencing data and microarray data have been submitted to GEO database. PMID:24143179

  19. The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics

    PubMed Central

    Bouchoux, Julien; Beilstein, Frauke; Pauquai, Thomas; Guerrera, I. Chiara; Chateau, Danielle; Ly, Nathalie; Alqub, Malik; Klein, Christophe; Chambaz, Jean; Rousset, Monique; Lacorte, Jean-Marc; Morel, Etienne; Demignot, Sylvie

    2011-01-01

    Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization. Results. Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has

  20. Genomic variants reveal differential evolutionary constraints on human transglutaminases and point towards unrecognized significance of transglutaminase 2

    PubMed Central

    Thangaraju, Kiruphagaran; Király, Róbert; Demény, Máté A.; András Mótyán, János; Fuxreiter, Mónika; Fésüs, László

    2017-01-01

    Transglutaminases (TGMs) catalyze Ca2+-dependent transamidation of proteins with specified roles in blood clotting (F13a) and in cornification (TGM1, TGM3). The ubiquitous TGM2 has well described enzymatic and non-enzymatic functions but in-spite of numerous studies its physiological function in humans has not been defined. We compared data on non-synonymous single nucleotide variations (nsSNVs) and loss-of-function variants on TGM1-7 and F13a from the Exome aggregation consortium dataset, and used computational and biochemical analysis to reveal the roles of damaging nsSNVs of TGM2. TGM2 and F13a display rarer damaging nsSNV sites than other TGMs and sequence of TGM2, F13a and TGM1 are evolutionary constrained. TGM2 nsSNVs are predicted to destabilize protein structure, influence Ca2+ and GTP regulation, and non-enzymatic interactions, but none coincide with conserved functional sites. We have experimentally characterized six TGM2 allelic variants detected so far in homozygous form, out of which only one, p.Arg222Gln, has decreased activities. Published exome sequencing data from various populations have not uncovered individuals with homozygous loss-of-function variants for TGM2, TGM3 and TGM7. Thus it can be concluded that human transglutaminases differ in harboring damaging variants and TGM2 is under purifying selection suggesting that it may have so far not revealed physiological functions. PMID:28248968

  1. Pattern Classification of Working Memory Networks Reveals Differential Effects of Methylphenidate, Atomoxetine, and Placebo in Healthy Volunteers

    PubMed Central

    Marquand, Andre F; De Simoni, Sara; O'Daly, Owen G; Williams, Steven CR; Mourão-Miranda, Janaina; Mehta, Mitul A

    2011-01-01

    Stimulant and non-stimulant drugs can reduce symptoms of attention deficit/hyperactivity disorder (ADHD). The stimulant drug methylphenidate (MPH) and the non-stimulant drug atomoxetine (ATX) are both widely used for ADHD treatment, but their differential effects on human brain function remain unclear. We combined event-related fMRI with multivariate pattern recognition to characterize the effects of MPH and ATX in healthy volunteers performing a rewarded working memory (WM) task. The effects of MPH and ATX on WM were strongly dependent on their behavioral context. During non-rewarded trials, only MPH could be discriminated from placebo (PLC), with MPH producing a similar activation pattern to reward. During rewarded trials both drugs produced the opposite effect to reward, that is, attenuating WM networks and enhancing task-related deactivations (TRDs) in regions consistent with the default mode network (DMN). The drugs could be directly discriminated during the delay component of rewarded trials: MPH produced greater activity in WM networks and ATX produced greater activity in the DMN. Our data provide evidence that: (1) MPH and ATX have prominent effects during rewarded WM in task-activated and -deactivated networks; (2) during the delay component of rewarded trials, MPH and ATX have opposing effects on activated and deactivated networks: MPH enhances TRDs more than ATX, whereas ATX attenuates WM networks more than MPH; and (3) MPH mimics reward during encoding. Thus, interactions between drug effects and motivational state are crucial in defining the effects of MPH and ATX. PMID:21346736

  2. Comparative Proteomic Analysis Reveals Differential Root Proteins in Medicago sativa and Medicago truncatula in Response to Salt Stress

    PubMed Central

    Long, Ruicai; Li, Mingna; Zhang, Tiejun; Kang, Junmei; Sun, Yan; Cong, Lili; Gao, Yanli; Liu, Fengqi; Yang, Qingchuan

    2016-01-01

    Salt stress is an important abiotic stress that causes decreased crop yields. Root growth and plant activities are affected by salt stress through the actions of specific genes that help roots adapt to adverse environmental conditions. For a more comprehensive understanding of proteins affected by salinity, we used two-dimensional gel electrophoresis and mass spectrometry to characterize the proteome-level changes associated with salt stress response in Medicago sativa cv. Zhongmu-1 and Medicago truncatula cv. Jemalong A17 roots. Our physiological and phenotypic observations indicated that Zhongmu-1 was more salt tolerant than Jemalong A17. We identified 93 and 30 proteins whose abundance was significantly affected by salt stress in Zhongmu-1 and Jemalong A17 roots, respectively. The tandem mass spectrometry analysis of the differentially accumulated proteins resulted in the identification of 60 and 26 proteins in Zhongmu-1 and Jemalong A17 roots, respectively. Function analyses indicated molecule binding and catalytic activity were the two primary functional categories. These proteins have known functions in various molecular processes, including defense against oxidative stress, metabolism, photosynthesis, protein synthesis and processing, and signal transduction. The transcript levels of four identified proteins were determined by quantitative reverse transcription polymerase chain reaction. Our results indicate that some of the identified proteins may play key roles in salt stress tolerance. PMID:27066057

  3. RNA sequencing reveals differential thermal regulation mechanisms between sexes of Glanville fritillary butterfly in the Tianshan Mountains, China.

    PubMed

    Lei, Ying; Wang, Yang; Ahola, Virpi; Luo, Shiqi; Xu, Chongren; Wang, Rongjiang

    2016-12-01

    The Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae) has been extensively studied as a model species in metapopulation ecology. We investigated in the earlier studies that female butterflies exhibit higher thermal tolerance than males in the Tianshan Mountains of China. We aim to understand the molecular mechanism of differences of thermal responses between sexes. We used RNA-seq approach and performed de novo assembly of transcriptome to compare the gene expression patterns between two sexes after heat stress. All the reads were assembled into 84,376 transcripts and 72,701 unigenes. The number of differential expressed genes (DEGs) between control and heat shock samples was 175 and 268 for males and females, respectively. Heat shock proteins genes (hsps) were up-regulated in response to heat stress in both males and females. Most of the up-regulated hsps showed higher fold changes in males than in females. Females expressed more ribosomal subunit protein genes, transcriptional elongation factor genes, and methionine-rich storage protein genes, participating in protein synthesis. It indicated that protein synthesis is needed for females to replace the damaged proteins due to heat shock. In addition, aspartate decarboxylase might contribute to thermal tolerance in females. These differences in gene expression may at least partly explain the response to high temperature stress, and the fact that females exhibit higher thermal tolerance.

  4. Differential expression of circadian clock genes in two strains of beetles reveals candidates related to photoperiodic induction of summer diapause.

    PubMed

    Zhu, Li; Liu, Wen; Tan, Qian-Qian; Lei, Chao-Liang; Wang, Xiao-Ping

    2017-03-01

    Diapause (also known as dormancy) is a state of arrested development induced by photoperiod or temperature that allows insects to survive adverse environmental conditions. By regulating diapause induction, the circadian clock is involved in short-day-induced winter diapause but whether this is also the case in long-day (LD)-induced summer diapause remains unknown. The cabbage beetle Colaphellus bowringi could enter summer diapause under LD conditions. However, a non-photoperiodic-diapause (NPD) strain of this species, which was developed in our laboratory by artificial selection, could not enter diapause under LD photoperiod. Therefore, we identified circadian clock genes in this species and measured differences in their expression between a high diapause (HD) strain and the NPD strain to investigate the potential relationship between circadian clock genes and summer diapause induction in C. bowringi. We successfully cloned eight circadian clock genes and obtained intact ORFs of four; cryptochrome2, double-time, shaggy and vrille. Phylogenetic trees and sequence alignment analyses indicated that these circadian clock genes were conserved across insect taxa. The quantitative real-time PCR indicated that clock, cycle, period, timeless, cryptochrome2, and vrille were differentially expressed between HD and NPD strains reared under LD photoperiod during the diapause induction phase. These findings suggest the potential relationship between circadian clock genes and LD-regulated summer diapause induction in C. bowringi.

  5. Landscape genomics and a common garden trial reveal adaptive differentiation to temperature across Europe in the tree species Alnus glutinosa.

    PubMed

    De Kort, Hanne; Vandepitte, Katrien; Bruun, Hans Henrik; Closset-Kopp, Déborah; Honnay, Olivier; Mergeay, Joachim

    2014-10-01

    The adaptive potential of tree species to cope with climate change has important ecological and economic implications. Many temperate tree species experience a wide range of environmental conditions, suggesting high adaptability to new environmental conditions. We investigated adaptation to regional climate in the drought-sensitive tree species Alnus glutinosa (Black alder), using a complementary approach that integrates genomic, phenotypic and landscape data. A total of 24 European populations were studied in a common garden and through landscape genomic approaches. Genotyping-by-sequencing was used to identify SNPs across the genome, resulting in 1990 SNPs. Although a relatively low percentage of putative adaptive SNPs was detected (2.86% outlier SNPs), we observed clear associations among outlier allele frequencies, temperature and plant traits. In line with the typical drought avoiding nature of A. glutinosa, leaf size varied according to a temperature gradient and significant associations with multiple outlier loci were observed, corroborating the ecological relevance of the observed outlier SNPs. Moreover, the lack of isolation by distance, the very low genetic differentiation among populations and the high intrapopulation genetic variation all support the notion that high gene exchange combined with strong environmental selection promotes adaptation to environmental cues.

  6. Comparative Proteomic Analysis Reveals Differential Root Proteins in Medicago sativa and Medicago truncatula in Response to Salt Stress.

    PubMed

    Long, Ruicai; Li, Mingna; Zhang, Tiejun; Kang, Junmei; Sun, Yan; Cong, Lili; Gao, Yanli; Liu, Fengqi; Yang, Qingchuan

    2016-01-01

    Salt stress is an important abiotic stress that causes decreased crop yields. Root growth and plant activities are affected by salt stress through the actions of specific genes that help roots adapt to adverse environmental conditions. For a more comprehensive understanding of proteins affected by salinity, we used two-dimensional gel electrophoresis and mass spectrometry to characterize the proteome-level changes associated with salt stress response in Medicago sativa cv. Zhongmu-1 and Medicago truncatula cv. Jemalong A17 roots. Our physiological and phenotypic observations indicated that Zhongmu-1 was more salt tolerant than Jemalong A17. We identified 93 and 30 proteins whose abundance was significantly affected by salt stress in Zhongmu-1 and Jemalong A17 roots, respectively. The tandem mass spectrometry analysis of the differentially accumulated proteins resulted in the identification of 60 and 26 proteins in Zhongmu-1 and Jemalong A17 roots, respectively. Function analyses indicated molecule binding and catalytic activity were the two primary functional categories. These proteins have known functions in various molecular processes, including defense against oxidative stress, metabolism, photosynthesis, protein synthesis and processing, and signal transduction. The transcript levels of four identified proteins were determined by quantitative reverse transcription polymerase chain reaction. Our results indicate that some of the identified proteins may play key roles in salt stress tolerance.

  7. Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

    2015-05-01

    Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

  8. Rab13 Is Upregulated During Osteoclast Differentiation and Associates with Small Vesicles Revealing Polarized Distribution in Resorbing Cells

    PubMed Central

    Mulari, Mika T. K.; Büki, Kálmán G.; Vihko, Pirkko; Härkönen, Pirkko L.; Väänänen, H. Kalervo

    2012-01-01

    Osteoclasts are bone-resorbing multinucleated cells that undergo drastic changes in their polarization due to heavy vesicular trafficking during the resorption cycle. These events require the precise orchestration of membrane traffic in order to maintain the unique characteristics of the different membrane domains in osteoclasts. Rab proteins are small GTPases involved in regulation of most, if not all, steps of vesicle trafficking. The investigators studied RAB genes in human osteoclasts and found that at least 26 RABs were expressed in osteoclasts. Out of these, RAB13 gene expression was highly upregulated during differentiation of human peripheral blood monocytic cells into osteoclasts. To study its possible function in osteoclasts, the investigators performed immunolocalization studies for Rab13 and various known markers of osteoclast vesicular trafficking. Rab13 localized to small vesicular structures at the superior parts of the osteoclast between the trans-Golgi network and basolateral membrane domain. Rab13 localization suggests that it is not involved in endocytosis or transcytosis of bone degradation products. In addition, Rab13 did not associate with early endosomes or recycling endosomes labeled with EEA1 or TRITC-conjugated transferrin, respectively. Its involvement in glucose transporter traffic was excluded as well. It is suggested that Rab13 is associated with a putative secretory function in osteoclasts. PMID:22562557

  9. Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging

    PubMed Central

    2014-01-01

    The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054. PMID:25181601

  10. Genetic differentiation of Octopus minor (Mollusca, Cephalopoda) off the northern coast of China as revealed by amplified fragment length polymorphisms.

    PubMed

    Yang, J M; Sun, G H; Zheng, X D; Ren, L H; Wang, W J; Li, G R; Sun, B C

    2015-12-02

    Octopus minor (Sasaki, 1920) is an economically important cephalopod that is found in the northern coastal waters of China. In this study, we investigated genetic differentiation in fishery populations using amplified fragment length polymorphisms (AFLPs). A total of 150 individuals were collected from five locations: Dalian (DL), Yan-tai (YT), Qingdao (QD), Lianyungang (LY), and Zhoushan (ZS), and 243 reproducible bands were amplified using five AFLP primer combinations. The percentage of polymorphic bands ranged from 53.33 to 76.08%. Nei's genetic identity ranged from 0.9139 to 0.9713, and the genetic distance ranged from 0.0291 to 0.0900. A phylogenetic tree was constructed using the unweighted pair group method with arithmetic mean, based on the genetic distance. The DL and YT populations originated from one clade, while the QD, LY, and ZS populations originated from another. The results indicate that the O. minor stock consisted of two genetic populations with an overall significantly analogous FST value (0.1088, P < 0.05). Most of the variance was within populations. These findings will be important for more sustainable octopus fisheries, so that this marine resource can be conserved for its long-term utilization.

  11. Differential adaptive responses to 1- or 2-day fasting in various mouse tissues revealed by quantitative PCR analysis

    PubMed Central

    Yamamoto, Junya; Kamata, Shotaro; Miura, Asumi; Nagata, Tomoko; Kainuma, Ryo; Ishii, Isao

    2015-01-01

    Dietary or caloric restriction confers various clinical benefits. Short-term fasting of mice is a common experimental procedure that may involve systemic metabolic remodeling, which may significantly affect experimental outputs. This study evaluated adaptive cellular responses after 1- or 2-day fasting in 13 mouse tissues by quantitative PCR using 15 marker primer sets for the activation of ubiquitin–proteasome (Atrogin-1 and MuRF1), autophagy–lysosome (LC3b, p62 and Lamp2), amino acid response (Asns, Trib3, Herpud1, xCT, and Chop), Nrf2-mediated antioxidant (HO-1 and Gsta1), and amino acid transport (Slc38a2, Slc7a5, and Slc7a1) systems. Differential activation profiles obtained in seven highly (thymus, liver, spleen, and small intestine) or mildly (stomach, kidney, and colon) atrophied tissues as well as in six non-atrophied tissues (brain, eye, lung, heart, skeletal muscle, and testis) suggested tissue-specific active metabolic remodeling. PMID:25973363

  12. EPG Recordings Reveal Differential Feeding Behaviors in Sogatella furcifera in Response to Plant Virus Infection and Transmission Success

    PubMed Central

    Lei, Wenbin; Li, Pei; Han, Yongqiang; Gong, Shaolong; Yang, Lang; Hou, Maolin

    2016-01-01

    Plant viruses are primarily transmitted by insect vectors and virus infection may influence on the vectors’ feeding behaviors. Using an electrical penetration graph, we detected that infection with the Southern rice black-streaked dwarf virus (SRBSDV) in the white-backed planthopper (WBPH) and in rice plants both altered the vector’s feeding behavior. When viruliferous WBPH (carrying SRBSDV) were fed on uninfected plants, they spent more time in salivation and phloem sap ingestion than non-viruliferous insects. In comparison with uninfected plants, infected plants showed an arrestant effect on non-viruliferous WBPH for phloem sap ingestion. Differential feeding behaviors were also detected between the WBPH that inoculated or acquired SRBSDV and those that failed to. The WBPH that inoculated SRBSDV exhibited more probing bouts, salivation events and phloem sap ingestion events and longer salivation than those that failed to. The WBPH that acquired SRBSDV were quicker to reach phloem and spent more time in phloem sap ingestion than those that failed to. These behavior alterations in the vector may have adaptive advantages for SRBSDV transmission and spread success because greater salivation by viruliferous vectors on uninfected hosts will promote virus inoculation, whereas more sap ingestion by non-viruliferous vectors on infected hosts will promote virus acquisition. PMID:27492995

  13. Suppression subtraction hybridization (SSH) and macroarray techniques reveal differential gene expression profiles in brain of sea bream infected with nodavirus.

    PubMed

    Dios, S; Poisa-Beiro, L; Figueras, A; Novoa, B

    2007-03-01

    Despite of the impact that viruses have on aquatic organisms, relatively little is known on how fish fight against these infections. In this work, the brain gene expression pattern of sea bream (Sparus aurata) in response to nodavirus infection was investigated. We used the suppression subtractive hybridization (SSH) method to generate a subtracted cDNA library enriched with gene transcripts differentially expressed after 1 day post-infection. Some of the ESTs from the infected tissues fell in gene categories related to stress and immune responses. For the reverse library (ESTs expressed in controls compared with infected tissues) the most abundant transcripts were of ribosomal and mitochondrial nature. Several ESTs potentially induced by virus exposure were selected for in vivo expression studies. We observed a clear difference in expression between infected and control samples for two candidate genes, ubiquitin conjugating enzyme 7 interacting protein, which seems to play an important role in apoptosis and the interferon induced protein with helicase C domain 1 (mda-5) that contributes to apoptosis and regulates the type I IFN production, a key molecule of the antiviral innate response in most organisms.

  14. High-throughput sequencing reveals differential regulation of miRNAs in fenoxaprop-P-ethyl-resistant Beckmannia syzigachne

    PubMed Central

    Pan, Lang; Wang, Zhaoyun; Cai, Jia; Gao, Haitao; Zhao, Hongwei; Dong, Liyao

    2016-01-01

    Non-target site resistance (NTSR) to herbicides is an increasing concern for weed control. The majority of previous studies have focused on metabolic resistance mechanisms of NTSR, but no research exists on gene regulation mechanisms behind herbicide resistance, such as microRNA (miRNA). Here, we identified 3 American sloughgrass (Beckmannia syzigachne Steud.) populations containing fenoxaprop-P-ethyl-resistant plants. We then constructed small RNA libraries and subjected them to deep sequencing and bioinformatics analyses. Forty known and 36 potentially novel, predicted miRNAs were successfully identified. Of these, we identified 3 conserved, predicted candidate NTSR-determinant miRNAs and their potential corresponding target genes, as well as 4 novel potential miRNAs with high count. Target gene prediction and annotation indicated that these 7 differentially expressed miRNAs potentially play a role in regulating specific stress-responsive genes, very likely related to herbicide resistance. Expression profiles were determined with quantitative real-time PCR. The present study is a novel, large-scale characterization of weed miRNAs. The results should further our understanding of miRNA expression profiles associated with herbicide resistance, allowing for the development of more effective weed management strategies. PMID:27353151

  15. Differential cysteine labeling and global label-free proteomics reveals an altered metabolic state in skeletal muscle aging.

    PubMed

    McDonagh, Brian; Sakellariou, Giorgos K; Smith, Neil T; Brownridge, Philip; Jackson, Malcolm J

    2014-11-07

    The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054.

  16. Proteomic responses reveal the differential effects induced by cadmium in mussels Mytilus galloprovincialis at early life stages.

    PubMed

    Xu, Lanlan; Peng, Xiao; Yu, Deliang; Ji, Chenglong; Zhao, Jianmin; Wu, Huifeng

    2016-08-01

    Cadmium (Cd) has become an important metal contaminant and posed severe risk on the organisms in the coastal environments of the Bohai Sea. Marine mussel Mytilus galloprovincialis is widely distributed along the Bohai coast and consumed as seafood by local residents. Evidences indicate that the early stages of marine organisms are more sensitive to metal contaminants. In this study, we applied two-dimensional electrophoresis-based proteomics to characterize the biological effects of Cd (50 μg L(-1)) in the early life stages (D-shape larval and juvenile) of mussels. The different proteomic responses demonstrated the differential responsive mechanisms to Cd exposure in these two early life stages of mussels. In details, results indicated that Cd mainly induced immune and oxidative stresses in both D-shape larval and juvenile mussels via different pathways. In addition, the significant up-regulation of triosephosphate isomerase and metallothionein confirmed the enhanced energy demand and mobilized detoxification mechanism in D-shape larval mussels exposed to Cd. In juvenile mussels, Cd exposure also induced clear apoptosis. Overall, this work suggests that Cd is a potential immune toxicant to mussel M. galloprovincialis at early life stages.

  17. Differential neural responses to acupuncture revealed by MEG using wavelet-based time-frequency analysis: a pilot study.

    PubMed

    You, Youbo; Bai, Lijun; Dai, Ruwei; Xue, Ting; Zhong, Chongguang; Feng, Yuanyuan; Wang, Hu; Liu, Zhenyu; Tian, Jie

    2011-01-01

    Acupoint specificity, lying at the core of the Traditional Chinese Medicine, still faces many controversies. As previous neuroimaging studies on acupuncture mainly adopted relatively low time-resolution functional magnetic resonance imaging (fMRI) technology and inappropriate block-designed experimental paradigm due to sustained effect, in the current study, we employed a single block-designed paradigm together with high temporal-resolution magnetoencephalography (MEG) technology. We applied time-frequency analysis based upon Morlet wavelet transforming approach to detect differential oscillatory brain dynamics induced by acupuncture at Stomach Meridian 36 (ST36) using a nearby nonacupoint (NAP) as control condition. We observed that frequency power changes were mainly restricted to delta band for both ST36 group and NAP group. Consistently increased delta band power in contralateral temporal regions and decreased power in the counterparts of ipsilateral hemisphere were detected following stimulation at ST36 on the right leg. Compared with ST36, no significant delta ranges were found in temporal regions in NAP group, illustrating different oscillatory brain patterns. Our results may provide additional evidence to support the specificity of acupuncture modulation effects.

  18. Inactivation of Ribosomal Protein Genes in Bacillus subtilis Reveals Importance of Each Ribosomal Protein for Cell Proliferation and Cell Differentiation

    PubMed Central

    Akanuma, Genki; Nanamiya, Hideaki; Natori, Yousuke; Yano, Koichi; Suzuki, Shota; Omata, Shuya; Ishizuka, Morio; Sekine, Yasuhiko

    2012-01-01

    Among the 57 genes that encode ribosomal proteins in the genome of Bacillus subtilis, a Gram-positive bacterium, 50 genes were targeted by systematic inactivation. Individual deletion mutants of 16 ribosomal proteins (L1, L9, L15, L22, L23, L28, L29, L32, L33.1, L33.2, L34, L35, L36, S6, S20, and S21) were obtained successfully. In conjunction with previous reports, 22 ribosomal proteins have been shown to be nonessential in B. subtilis, at least for cell proliferation. Although several mutants that harbored a deletion of a ribosomal protein gene did not show any significant differences in any of the phenotypes that were tested, various mutants showed a reduced growth rate and reduced levels of 70S ribosomes compared with the wild type. In addition, severe defects in the sporulation frequency of the ΔrplA (L1) mutant and the motility of the ΔrpsU (S21) mutant were observed. These data provide the first evidence in B. subtilis that L1 and S21 are required for the progression of cellular differentiation. PMID:23002217

  19. A Populus TIR1 gene family survey reveals differential expression patterns and responses to 1-naphthaleneacetic acid and stress treatments

    PubMed Central

    Shu, Wenbo; Liu, Yingli; Guo, Yinghua; Zhou, Houjun; Zhang, Jin; Zhao, Shutang; Lu, Mengzhu

    2015-01-01

    The plant hormone auxin is a central regulator of plant growth. TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) is a component of the E3 ubiquitin ligase complex SCFTIR1/AFB and acts as an auxin co-receptor for nuclear auxin signaling. The SCFTIR1/AFB-proteasome machinery plays a central regulatory role in development-related gene transcription. Populus trichocarpa, as a model tree, has a unique fast-growth trait to which auxin signaling may contribute. However, no systematic analyses of the genome organization, gene structure, and expression of TIR1-like genes have been undertaken in this woody model plant. In this study, we identified a total of eight TIR1 genes in the Populus genome that are phylogenetically clustered into four subgroups, PtrFBL1/PtrFBL2, PtrFBL3/PtrFBL4, PtrFBL5/PtrFBL6, and PtrFBL7/PtrFBL8, representing four paralogous pairs. In addition, the gene structure and motif composition were relatively conserved in each paralogous pair and all of the PtrFBL members were localized in the nucleus. Different sets of PtrFBLs were strongly expressed in the leaves, stems, roots, cambial zones, and immature xylem of Populus. Interestingly, PtrFBL1 and 7 were expressed mainly in vascular and cambial tissues, respectively, indicating their potential but different roles in wood formation. Furthermore, Populus FBLs responded differentially upon exposure to various stresses. Finally, over-expression studies indicated a role of FBL1 in poplar stem growth and response to drought stress. Collectively, these observations lay the foundation for further investigations into the potential roles of PtrFBL genes in tree growth and development. PMID:26442033

  20. Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages

    PubMed Central

    Rao, Xiaolan; Lu, Nan; Li, Guifen; Nakashima, Jin; Tang, Yuhong; Dixon, Richard A.

    2016-01-01

    Almost all C4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C4 photosynthesis in the two cell types within C4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C4 photosynthesis, while distinct C4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C4 species. PMID:26896851

  1. Transcript Quantification by RNA-Seq Reveals Differentially Expressed Genes in the Red and Yellow Fruits of Fragaria vesca.

    PubMed

    Zhang, Yuchao; Li, Weijia; Dou, Yujuan; Zhang, Junxiang; Jiang, Guihua; Miao, Lixiang; Han, Guofen; Liu, Yuexue; Li, He; Zhang, Zhihong

    2015-01-01

    Fragaria vesca (2n = 2x = 14), the woodland strawberry, is a perennial herbaceous plant with a small sequenced genome (240 Mb). It is commonly used as a genetic model plant for the Fragaria genus and the Rosaceae family. Fruit skin color is one of the most important traits for both the commercial and esthetic value of strawberry. Anthocyanins are the most prominent pigments in strawberry that bring red, pink, white, and yellow hues to the fruits in which they accumulate. In this study, we conducted a de novo assembly of the fruit transcriptome of woodland strawberry and compared the gene expression profiles with yellow (Yellow Wonder, YW) and red (Ruegen, RG) fruits. De novo assembly yielded 75,426 unigenes, 21.3% of which were longer than 1,000 bp. Among the high-quality unique sequences, 45,387 (60.2%) had at least one significant match to an existing gene model. A total of 595 genes, representing 0.79% of total unigenes, were differentially expressed in YW and RG. Among them, 224 genes were up-regulated and 371 genes were down-regulated in the fruit of YW. Particularly, some flavonoid biosynthetic pathway genes, including C4H, CHS, CHI, F3H, DFR and ANS, as well as some transcription factors (TFs), including MYB (putative MYB86 and MYB39), WDR and MADS, were down-regulated in YW fruit, concurrent with a reduction in anthocyanin accumulation in the yellow pigment phenotype, whereas a putative transcription repressor MYB1R was up-regulated in YW fruit. The altered expression levels of the genes encoding flavonoid biosynthetic enzymes and TFs were confirmed by quantitative RT-PCR. Our study provides important insights into the molecular mechanisms underlying the yellow pigment phenotype in F. vesca.

  2. Differentiation State-Specific Mitochondrial Dynamic Regulatory Networks Are Revealed by Global Transcriptional Analysis of the Developing Chicken Lens

    PubMed Central

    Chauss, Daniel; Basu, Subhasree; Rajakaruna, Suren; Ma, Zhiwei; Gau, Victoria; Anastas, Sara; Brennan, Lisa A.; Hejtmancik, J. Fielding; Menko, A. Sue; Kantorow, Marc

    2014-01-01

    The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that requires a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected more than 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, more than 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic (DUT, PDK1, SNPH), autophagy (ATG3, ATG4B, BECN1, FYCO1, WIPI1), and mitophagy (BNIP3L/NIX, BNIP3, PARK2, p62/SQSTM1) transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells. PMID:24928582

  3. Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages.

    PubMed

    Rao, Xiaolan; Lu, Nan; Li, Guifen; Nakashima, Jin; Tang, Yuhong; Dixon, Richard A

    2016-03-01

    Almost all C4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C4 photosynthesis in the two cell types within C4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C4 photosynthesis, while distinct C4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C4 species.

  4. Transcriptome Analysis of Blunt Snout Bream (Megalobrama amblycephala) Reveals Putative Differential Expression Genes Related to Growth and Hypoxia

    PubMed Central

    Li, Fu-Gui; Chen, Jie; Jiang, Xia-Yun; Zou, Shu-Ming

    2015-01-01

    The blunt snout bream (Megalobrama amblycephala) is an important freshwater aquaculture species, but it is sensitive to hypoxia. No transcriptome data related to growth and hypoxia response are available for this species. In this study, we performed de novo transcriptome sequencing for the liver and gills of the fast-growth family and slow-growth family derived from ‘Pujiang No.1’ F10 blunt snout bream that were under hypoxic stress and normoxia, respectively. The fish were divided into the following 4 groups: fast-growth family under hypoxic stress, FH; slow-growth family under hypoxic stress, SH; fast-growth family under normoxia, FN; and slow-growth family under normoxia, SN. A total of 185 million high-quality reads were obtained from the normalized cDNA of the pooled samples, which were assembled into 465,582 contigs and 237,172 transcripts. A total of 31,338 transcripts from the same locus (unigenes) were annotated and assigned to 104 functional groups, and 23,103 unigenes were classified into seven main categories, including 45 secondary KEGG pathways. A total of 22,255 (71%) known putative unigenes were found to be shared across the genomes of five model fish species and mammals, and a substantial number (9.4%) of potentially novel genes were identified. When 6,639 unigenes were used in the analysis of differential expression (DE) genes, the number of putative DE genes related to growth pathways in FH, SH, SN and FN was 159, 118, 92 and 65 in both the liver and gills, respectively, and the number of DE genes related to hypoxic response was 57, 33, 23 and 21 in FH, FN, SH and SN, respectively. Our results suggest that growth performance of the fast-growth family should be due to complex mutual gene regulatory mechanisms of these putative DE genes between growth and hypoxia. PMID:26554582

  5. Lineage tracing of neuromesodermal progenitors reveals novel Wnt-dependent roles in trunk progenitor cell maintenance and differentiation.

    PubMed

    Garriock, Robert J; Chalamalasetty, Ravindra B; Kennedy, Mark W; Canizales, Lauren C; Lewandoski, Mark; Yamaguchi, Terry P

    2015-05-01

    In the development of the vertebrate body plan, Wnt3a is thought to promote the formation of paraxial mesodermal progenitors (PMPs) of the trunk region while suppressing neural specification. Recent lineage-tracing experiments have demonstrated that these trunk neural progenitors and PMPs derive from a common multipotent progenitor called the neuromesodermal progenitor (NMP). NMPs are known to reside in the anterior primitive streak (PS) region; however, the extent to which NMPs populate the PS and contribute to the vertebrate body plan, and the precise role that Wnt3a plays in regulating NMP self-renewal and differentiation are unclear. To address this, we used cell-specific markers (Sox2 and T) and tamoxifen-induced Cre recombinase-based lineage tracing to locate putative NMPs in vivo. We provide functional evidence for NMP location primarily in the epithelial PS, and to a lesser degree in the ingressed PS. Lineage-tracing studies in Wnt3a/β-catenin signaling pathway mutants provide genetic evidence that trunk progenitors normally fated to enter the mesodermal germ layer can be redirected towards the neural lineage. These data, combined with previous PS lineage-tracing studies, support a model that epithelial anterior PS cells are Sox2(+)T(+) multipotent NMPs and form the bulk of neural progenitors and PMPs of the posterior trunk region. Finally, we find that Wnt3a/β-catenin signaling directs trunk progenitors towards PMP fates; however, our data also suggest that Wnt3a positively supports a progenitor state for both mesodermal and neural progenitors.

  6. Analysis of Drosophila p8 and p52 mutants reveals distinct roles for the maintenance of TFIIH stability and male germ cell differentiation

    PubMed Central

    Cruz-Becerra, Grisel; Juárez, Mandy; Valadez-Graham, Viviana

    2016-01-01

    Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. Here, we show that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, we found that TFIIH stability is not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients. PMID:27805905

  7. Atomic force microscopy reveals a morphological differentiation of chromobacterium violaceum cells associated with biofilm development and directed by N-hexanoyl-L-homoserine lactone.

    PubMed

    Kamaeva, Anara A; Vasilchenko, Alexey S; Deryabin, Dmitry G

    2014-01-01

    Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure.

  8. Mitochondrial DNA markers reveal high genetic diversity but low genetic differentiation in the black fly Simulium tani Takaoka & Davies along an elevational gradient in Malaysia.

    PubMed

    Low, Van Lun; Adler, Peter H; Takaoka, Hiroyuki; Ya'cob, Zubaidah; Lim, Phaik Eem; Tan, Tiong Kai; Lim, Yvonne A L; Chen, Chee Dhang; Norma-Rashid, Yusoff; Sofian-Azirun, Mohd

    2014-01-01

    The population genetic structure of Simulium tani was inferred from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) and II (COII) along an elevational gradient in Cameron Highlands, Malaysia. A statistical parsimony network of 71 individuals revealed 71 haplotypes in the COI gene and 43 haplotypes in the COII gene; the concatenated sequences of the COI and COII genes revealed 71 haplotypes. High levels of genetic diversity but low levels of genetic differentiation were observed among populations of S. tani at five elevations. The degree of genetic diversity, however, was not in accordance with an altitudinal gradient, and a Mantel test indicated that elevation did not have a limiting effect on gene flow. No ancestral haplotype of S. tani was found among the populations. Pupae with unique structural characters at the highest elevation showed a tendency to form their own haplotype cluster, as revealed by the COII gene. Tajima's D, Fu's Fs, and mismatch distribution tests revealed population expansion of S. tani in Cameron Highlands. A strong correlation was found between nucleotide diversity and the levels of dissolved oxygen in the streams where S. tani was collected.

  9. Genome‐wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation

    PubMed Central

    Pai, Vaibhav P.; Martyniuk, Christopher J.; Echeverri, Karen; Sundelacruz, Sarah; Kaplan, David L.

    2015-01-01

    Abstract Endogenous bioelectric signaling via changes in cellular resting potential (V mem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of V mem has been functionally implicated in dedifferentiation, tumorigenesis, anatomical re‐specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome‐wide transcriptional responses to V mem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to V mem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both subnetwork enrichment and PANTHER analyses identified a number of key genetic modules as targets of V mem change, and also revealed important (well‐conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm, and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that V mem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies. PMID:27499876

  10. Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation.

    PubMed

    Pai, Vaibhav P; Martyniuk, Christopher J; Echeverri, Karen; Sundelacruz, Sarah; Kaplan, David L; Levin, Michael

    2016-02-01

    Endogenous bioelectric signaling via changes in cellular resting potential (V mem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of V mem has been functionally implicated in dedifferentiation, tumorigenesis, anatomical re-specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome-wide transcriptional responses to V mem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to V mem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both subnetwork enrichment and PANTHER analyses identified a number of key genetic modules as targets of V mem change, and also revealed important (well-conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm, and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that V mem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies.

  11. Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development.

    PubMed Central

    Hansen, L. A.; Alexander, N.; Hogan, M. E.; Sundberg, J. P.; Dlugosz, A.; Threadgill, D. W.; Magnuson, T.; Yuspa, S. H.

    1997-01-01

    Mice harboring a targeted disruption of the epidermal growth factor receptor (EGFR) allele exhibit a severely disorganized hair follicle phenotype, fuzzy coat, and systemic disease resulting in death before 3 weeks. This skin phenotype was reproduced in whole skin grafts and in grafts of EGFR null hair follicle buds onto nude mice, providing a model to evaluate the natural evolution of skin lacking the EGFR. Hair follicles in grafts of null skin did not progress from anagen to telogen and scanning electron micrografts revealed wavy, flattened hair fibers with cuticular abnormalities. Many of the EGFR null hair follicles in the grafted skin were consumed by an inflammatory reaction resulting in complete hair loss in 67% of the grafts by 10 weeks. Localization of follicular differentiation markers including keratin 6, transglutaminase, and the hair keratins mHa2 and hacl-1 revealed a pattern of premature differentiation within the null hair follicles. In intact EGFR null mice, proliferation in the interfollicular epidermis, but not hair follicles, was greatly decreased in the absence of EGFR. In contrast, grafting of EGFR null skin resulted in a hyperplastic response in the epidermis that did not resolve even after 10 weeks, although the wound-induced hyperplasia in EGFR wild-type grafts had resolved within 3 to 4 weeks. Thus, epithelial expression of the EGFR has complex functions in the skin. It is important in delaying follicular differentiation, may serve to protect the hair follicle from immunological reactions, and modifies both normal and wound-induced epidermal proliferation but seems dispensable for follicular proliferation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:9176390

  12. Differential Responses to a Visual Self-Motion Signal in Human Medial Cortical Regions Revealed by Wide-View Stimulation

    PubMed Central

    Wada, Atsushi; Sakano, Yuichi; Ando, Hiroshi

    2016-01-01

    Vision is important for estimating self-motion, which is thought to involve optic-flow processing. Here, we investigated the fMRI response profiles in visual area V6, the precuneus motion area (PcM), and the cingulate sulcus visual area (CSv)—three medial brain regions recently shown to be sensitive to optic-flow. We used wide-view stereoscopic stimulation to induce robust self-motion processing. Stimuli included static, randomly moving, and coherently moving dots (simulating forward self-motion). We varied the stimulus size and the presence of stereoscopic information. A combination of univariate and multi-voxel pattern analyses (MVPA) revealed that fMRI responses in the three regions differed from each other. The univariate analysis identified optic-flow selectivity and an effect of stimulus size in V6, PcM, and CSv, among which only CSv showed a significantly lower response to random motion stimuli compared with static conditions. Furthermore, MVPA revealed an optic-flow specific multi-voxel pattern in the PcM and CSv, where the discrimination of coherent motion from both random motion and static conditions showed above-chance prediction accuracy, but that of random motion from static conditions did not. Additionally, while area V6 successfully classified different stimulus sizes regardless of motion pattern, this classification was only partial in PcM and was absent in CSv. This may reflect the known retinotopic representation in V6 and the absence of such clear visuospatial representation in CSv. We also found significant correlations between the strength of subjective self-motion and univariate activation in all examined regions except for primary visual cortex (V1). This neuro-perceptual correlation was significantly higher for V6, PcM, and CSv when compared with V1, and higher for CSv when compared with the visual motion area hMT+. Our convergent results suggest the significant involvement of CSv in self-motion processing, which may give rise to its

  13. Holistic microstructural techniques reveal synchronous and alternating andalusite and staurolite growth during three tectonic events resulted from shifting partitioning of growth vs deformation

    NASA Astrophysics Data System (ADS)

    Bell, T. H.; Fay, C.

    2016-10-01

    Excellent inclusion trails in a sample containing both staurolite and andalusite porphyroblasts are used to demonstrate techniques that allow the intimate relationships between deformation and porphyroblast growth to be recognized, described in detail and understood. This approach reveals three main phases of growth of both mineral phases, some of which was demonstrably synchronous, during three tectonic events. Each main period of growth occurred during the early stages of three deformations that were successively near orthogonal. However, extra periods are distinguishable in andalusite in some of these events because this phase occurs as clusters of large crystals that vary in orientation by 2° to > 10°. All foliations defined by all inclusion trails within every porphyroblast inflect/intersect about an axis trending at 025° (called a FIA). This indicates that the direction of the horizontal component of bulk shortening was identical for the first and third of the three deformations recorded by porphyroblast growth. Portions of sigmoidal to slightly spiral-shaped inclusion trails in most porphyroblast clusters locally diverge in opposite directions due to overprinting orthogonal bulk shortening typical of that which forms millipede geometries. These microstructures confirm the role of coaxial bulk shortening in initiating porphyroblast growth in an environment that locally becomes strongly non-coaxial as the deformation intensifies in the same event. In this sample, increasing non-coaxiality as the deformation intensified resulted in the same asymmetry for each of the three events and thus an overall spiral-like shape. Differing stages in the development of these bulk-shortening geometries preserved in adjacent or touching phases negate any role for porphyroblast rotation during ductile deformation. Andalusite and staurolite grew without any inter-reaction in locations where they lie in contact. This multiply repeated growth behaviour initiated within zones of

  14. Differential Dopamine Release Dynamics in the Nucleus Accumbens Core and Shell Reveal Complementary Signals for Error Prediction and Incentive Motivation

    PubMed Central

    Cacciapaglia, Fabio; Wightman, R. Mark; Carelli, Regina M.

    2015-01-01

    Mesolimbic dopamine (DA) is phasically released during appetitive behaviors, though there is substantive disagreement about the specific purpose of these DA signals. For example, prediction error (PE) models suggest a role of learning, while incentive salience (IS) models argue that the DA signal imbues stimuli with value and thereby stimulates motivated behavior. However, within the nucleus accumbens (NAc) patterns of DA release can strikingly differ between subregions, and as such, it is possible that these patterns differentially contribute to aspects of PE and IS. To assess this, we measured DA release in subregions of the NAc during a behavioral task that spatiotemporally separated sequential goal-directed stimuli. Electrochemical methods were used to measure subsecond NAc dopamine release in the core and shell during a well learned instrumental chain schedule in which rats were trained to press one lever (seeking; SL) to gain access to a second lever (taking; TL) linked with food delivery, and again during extinction. In the core, phasic DA release was greatest following initial SL presentation, but minimal for the subsequent TL and reward events. In contrast, phasic shell DA showed robust release at all task events. Signaling decreased between the beginning and end of sessions in the shell, but not core. During extinction, peak DA release in the core showed a graded decrease for the SL and pauses in release during omitted expected rewards, whereas shell DA release decreased predominantly during the TL. These release dynamics suggest parallel DA signals capable of supporting distinct theories of appetitive behavior. SIGNIFICANCE STATEMENT Dopamine signaling in the brain is important for a variety of cognitive functions, such as learning and motivation. Typically, it is assumed that a single dopamine signal is sufficient to support these cognitive functions, though competing theories disagree on how dopamine contributes to reward-based behaviors. Here, we have

  15. Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone

    PubMed Central

    Gottardini, Elena; Cristofori, Antonella; Pellegrini, Elisa; La Porta, Nicola; Nali, Cristina; Baldi, Paolo; Sablok, Gaurav

    2016-01-01

    Tropospheric ozone (O3) is a global air pollutant that causes high economic damages by decreasing plant productivity. It enters the leaves through the stomata, generates reactive oxygen species, which subsequent decrease in photosynthesis, plant growth, and biomass accumulation. In order to identify genes that are important for conferring O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic O3 treatment (60 ppb, 5 h d−1 for 45 consecutive days). Transcript profiling and relative expression assessment were carried out in asymptomatic leaves, after 15 days of O3 exposure. At the end of the experiment symptoms were observed on all treated leaves and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and relative expression assessment were carried out on sequenced reads. A total of 38,800 and 12,495 high quality reads obtained in control and O3-treated libraries, respectively (average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance was measured by reads per kilobase per million and 41 and 37 ensembl unigenes showed up- and down-regulation respectively. Unigenes functionally associated to photosynthesis and carbon utilization were repressed, demonstrating the deleterious effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts for several functional categories, including

  16. A transcriptomic study reveals differentially expressed genes and pathways respond to simulated acid rain in Arabidopsis thaliana.

    PubMed

    Liu, Ting-Wu; Niu, Li; Fu, Bin; Chen, Juan; Wu, Fei-Hua; Chen, Juan; Wang, Wen-Hua; Hu, Wen-Jun; He, Jun-Xian; Zheng, Hai-Lei

    2013-01-01

    Acid rain, as a worldwide environmental issue, can cause serious damage to plants. In this study, we provided the first case study on the systematic responses of arabidopsis (Arabidopsis thaliana (L.) Heynh.) to simulated acid rain (SiAR) by transcriptome approach. Transcriptomic analysis revealed that the expression of a set of genes related to primary metabolisms, including nitrogen, sulfur, amino acid, photosynthesis, and reactive oxygen species metabolism, were altered under SiAR. In addition, transport and signal transduction related pathways, especially calcium-related signaling pathways, were found to play important roles in the response of arabidopsis to SiAR stress. Further, we compared our data set with previously published data sets on arabidopsis transcriptome subjected to various stresses, including wound, salt, light, heavy metal, karrikin, temperature, osmosis, etc. The results showed that many genes were overlapped in several stresses, suggesting that plant response to SiAR is a complex process, which may require the participation of multiple defense-signaling pathways. The results of this study will help us gain further insights into the response mechanisms of plants to acid rain stress.

  17. Millennial-scale faunal record reveals differential resilience of European large mammals to human impacts across the Holocene.

    PubMed

    Crees, Jennifer J; Carbone, Chris; Sommer, Robert S; Benecke, Norbert; Turvey, Samuel T

    2016-03-30

    The use of short-term indicators for understanding patterns and processes of biodiversity loss can mask longer-term faunal responses to human pressures. We use an extensive database of approximately 18,700 mammalian zooarchaeological records for the last 11,700 years across Europe to reconstruct spatio-temporal dynamics of Holocene range change for 15 large-bodied mammal species. European mammals experienced protracted, non-congruent range losses, with significant declines starting in some species approximately 3000 years ago and continuing to the present, and with the timing, duration and magnitude of declines varying individually between species. Some European mammals became globally extinct during the Holocene, whereas others experienced limited or no significant range change. These findings demonstrate the relatively early onset of prehistoric human impacts on postglacial biodiversity, and mirror species-specific patterns of mammalian extinction during the Late Pleistocene. Herbivores experienced significantly greater declines than carnivores, revealing an important historical extinction filter that informs our understanding of relative resilience and vulnerability to human pressures for different taxa. We highlight the importance of large-scale, long-term datasets for understanding complex protracted extinction processes, although the dynamic pattern of progressive faunal depletion of European mammal assemblages across the Holocene challenges easy identification of 'static' past baselines to inform current-day environmental management and restoration.

  18. Simulating natural light and temperature cycles in the laboratory reveals differential effects on activity/rest rhythm of four Drosophilids.

    PubMed

    Prabhakaran, Priya M; Sheeba, Vasu

    2014-10-01

    Recent studies under semi-natural conditions have revealed various unique features of activity/rest rhythms in Drosophilids that differ from those under standard laboratory conditions. An additional afternoon peak (A-peak) has been reported for Drosophila melanogaster and another species D. malerkotliana while D. ananassae exhibited mostly unimodal diurnal activity. To tease apart the role of light and temperature in mediating these species-specific behaviours of four Drosophilid species D. melanogaster, D. malerkotliana, D. ananassae, and Zaprionus indianus we simulated gradual natural light and/or temperature cycles conditions in laboratory. The pattern observed under semi-natural conditions could be reproduced in the laboratory for all the species under a variety of simulated conditions. D. melanogaster and D. malerkotliana showed similar patterns where as D. ananassae consistently exhibited predominant morning activity under almost all regimes. Z. indianus showed clearly rhythmic activity mostly when temperature cycles were provided. We find that gradually changing light intensities reaching a sufficiently high peak value can elicit A-peak in D. melanogaster, D. malerkotliana, and D. ananassae even at mild ambient temperature. Furthermore, we show that high mid-day temperature could induce A-peak in all species even under constant light conditions suggesting that this A-peak is likely to be a stress response.

  19. Millennial-scale faunal record reveals differential resilience of European large mammals to human impacts across the Holocene

    PubMed Central

    Crees, Jennifer J.; Carbone, Chris; Sommer, Robert S.; Benecke, Norbert; Turvey, Samuel T.

    2016-01-01

    The use of short-term indicators for understanding patterns and processes of biodiversity loss can mask longer-term faunal responses to human pressures. We use an extensive database of approximately 18 700 mammalian zooarchaeological records for the last 11 700 years across Europe to reconstruct spatio-temporal dynamics of Holocene range change for 15 large-bodied mammal species. European mammals experienced protracted, non-congruent range losses, with significant declines starting in some species approximately 3000 years ago and continuing to the present, and with the timing, duration and magnitude of declines varying individually between species. Some European mammals became globally extinct during the Holocene, whereas others experienced limited or no significant range change. These findings demonstrate the relatively early onset of prehistoric human impacts on postglacial biodiversity, and mirror species-specific patterns of mammalian extinction during the Late Pleistocene. Herbivores experienced significantly greater declines than carnivores, revealing an important historical extinction filter that informs our understanding of relative resilience and vulnerability to human pressures for different taxa. We highlight the importance of large-scale, long-term datasets for understanding complex protracted extinction processes, although the dynamic pattern of progressive faunal depletion of European mammal assemblages across the Holocene challenges easy identification of ‘static’ past baselines to inform current-day environmental management and restoration. PMID:27009229

  20. Genome sequence comparison reveals a candidate gene involved in male-hermaphrodite differentiation in papaya (Carica papaya) trees.

    PubMed

    Ueno, Hiroki; Urasaki, Naoya; Natsume, Satoshi; Yoshida, Kentaro; Tarora, Kazuhiko; Shudo, Ayano; Terauchi, Ryohei; Matsumura, Hideo

    2015-04-01

    The sex type of papaya (Carica papaya) is determined by the pair of sex chromosomes (XX, female; XY, male; and XY(h), hermaphrodite), in which there is a non-recombining genomic region in the Y and Y(h) chromosomes. This region is presumed to be involved in determination of males and hermaphrodites; it is designated as the male-specific region in the Y chromosome (MSY) and the hermaphrodite-specific region in the Y(h) chromosome (HSY). Here, we identified the genes determining male and hermaphrodite sex types by comparing MSY and HSY genomic sequences. In the MSY and HSY genomic regions, we identified 14,528 nucleotide substitutions and 965 short indels with a large gap and two highly diverged regions. In the predicted genes expressed in flower buds, we found no nucleotide differences leading to amino acid changes between the MSY and HSY. However, we found an HSY-specific transposon insertion in a gene (SVP like) showing a similarity to the Short Vegetative Phase (SVP) gene. Study of SVP-like transcripts revealed that the MSY allele encoded an intact protein, while the HSY allele encoded a truncated protein. Our findings demonstrated that the SVP-like gene is a candidate gene for male-hermaphrodite determination in papaya.

  1. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.

    PubMed

    Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, HyungWon; Sobreira, Tiago J P; Nohara, Lilian L; Wermelinger, Luciana S; Almeida, Igor C; Zingali, Russolina B; Fernandes, Patricia M B

    2012-06-18

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses.

  2. Quantitative lid dynamics of MDM2 reveals differential ligand binding modes of the p53-binding cleft.

    PubMed

    Showalter, Scott A; Bruschweiler-Li, Lei; Johnson, Eric; Zhang, Fengli; Brüschweiler, Rafael

    2008-05-21

    The oncoprotein MDM2 regulates the activity and stability of the tumor suppressor p53 through protein-protein interaction involving their N-terminal domains. The N-terminal lid of MDM2 has been implicated in p53 regulation; however, due to its flexible nature, limited data are available concerning its role in ligand binding. The quantitative dynamics study using NMR reported here shows, for the first time, that the lid in apo-MDM2 slowly interconverts between a "closed" state that is associated with the p53-binding cleft and an "open" state that is highly flexible. Our results reveal that apo-MDM2 predominantly populates the closed state, whereas the p53-bound MDM2 exclusively populates the open state. Unlike p53 binding, the small molecule MDM2 antagonist nutlin-3 binds to the cleft essentially without perturbing the closed lid state. The lid dynamics thereby represents a signature for the experimental and virtual screening of therapeutic antagonists that target the p53-MDM2 interaction.

  3. Alternate cadmium exposure differentially affects the content of gamma-aminobutyric acid (GABA) and taurine within the hypothalamus, median eminence, striatum and prefrontal cortex of male rats.

    PubMed

    Esquifino, A I; Seara, R; Fernández-Rey, E; Lafuente, A

    2001-05-01

    This work examines changes of gamma aminobutyric acid (GABA) and taurine contents in the hypothalamus, striatum and prefrontal cortex of the rat after an alternate schedule of cadmium administration. Age-associated changes were also evaluated, of those before puberty and after adult age. In control rats GABA content decreased with age in the median eminence and in anterior, mediobasal and posterior hypothalamus, prefrontal cortex and the striatum. Taurine content showed similar results with the exception of mediobasal hypothalamus and striatum, where no changes were detected. In pubertal rats treated with cadmium from 30 to 60 days of life, GABA content significantly decreased in all brain regions except in the striatum. When cadmium was administered from day 60 to 90 of life, GABA content was significantly changed in prefrontal cortex only compared with the age matched controls. Taurine content showed similar results in pubertal rats, with the exception of the median eminence and the mediobasal hypothalamus, neither of which showed a change. However, when cadmium was administered to rats from day 60 to 90 of life, taurine content only changed in prefrontal cortex compared with the age matched controls. These results suggest that cadmium differentially affects GABA and taurine contents within the hypothalamus, median eminence, striatum and prefrontal cortex as a function of age.

  4. Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress

    PubMed Central

    Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

    2017-01-01

    Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress. DOI: http://dx.doi.org/10.7554/eLife.21907.001 PMID:28177284

  5. Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

    PubMed

    Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

    2017-02-08

    Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

  6. Differential Communications between Fungi and Host Plants Revealed by Secretome Analysis of Phylogenetically Related Endophytic and Pathogenic Fungi

    PubMed Central

    Xu, Xihui; He, Qin; Zhang, Chulong

    2016-01-01

    During infection, both phytopathogenic and endophytic fungi form intimate contact with living plant cells, and need to resist or disable host defences and modify host metabolism to adapt to their host. Fungi can achieve these changes by secreting proteins and enzymes. A comprehensive comparison of the secretomes of both endophytic and pathogenic fungi can improve our understanding of the interactions between plants and fungi. Although Magnaporthe oryzae, Gaeumannomyces graminis, and M. poae are economically important fungal pathogens, and the related species Harpophora oryzae is an endophyte, they evolved from a common pathogenic ancestor. We used a pipeline analysis to predict the H. oryzae, M. oryzae, G. graminis, and M. poae secretomes and identified 1142, 1370, 1001, and 974 proteins, respectively. Orthologue gene analyses demonstrated that the M. oryzae secretome evolved more rapidly than those of the other three related species, resulting in many species-specific secreted protein-encoding genes, such as avirulence genes. Functional analyses highlighted the abundance of proteins involved in the breakdown of host plant cell walls and oxidation-reduction processes. We identified three novel motifs in the H. and M. oryzae secretomes, which may play key roles in the interaction between rice and H. oryzae. Furthermore, we found that expression of the H. oryzae secretome involved in plant cell wall degradation was downregulated, but the M. oryzae secretome was upregulated with many more upregulated genes involved in oxidation-reduction processes. The divergent in planta expression patterns of the H. and M. oryzae secretomes reveal differences that are associated with mutualistic and pathogenic interactions, respectively. PMID:27658302

  7. Metabolomic profiling reveals differential effects of glucagon-like peptide-1 and insulin on nutrient partitioning in ovine liver.

    PubMed

    El-Sabagh, Mabrouk; Taniguchi, Dai; Sugino, Toshihisa; Obitsu, Taketo

    2016-12-01

    This study was conducted to identify the insulin-independent actions of glucagon-like peptide-1 (GLP-1 (7-36 amide)) in partitioning nutrient metabolism in ovine liver. Four Suffolk wethers (60.0 ± 6.7 kg body weight (BW)) were used in a repeated-measure design under euglycemic--hyperinsulinemic and hyper -GLP-1 clamps for 150 min with intravenous infusion of insulin (0.5 mU/kg BW/min; from 0 to 90 min), GLP-1 (0.5 µg/kg BW/min; from 60 to 150 min) and both hormones co-administered from 60 to 90 min. Liver biopsies were collected at 0, 60, 90 and 150 min to represent the metabolomic profiling of baseline, insulin, insulin plus GLP-1, and GLP-1, respectively, and were analyzed for metabolites using Capillary Electrophoresis Time-of-Flight Mass Spectrometer. Metabolomics analysis reveals 51 metabolites as being significantly altered (P < 0.05) by insulin and GLP-1 infusion compared to baseline values. Insulin infusion enhanced glycolysis, lipogenesis, oxidative stress defense and cell proliferation pathways, but reduced protein breakdown, gluconeogenesis and ketogenesis pathways. Conversely, GLP-1 infusion promoted lipolytic and ketogenic pathways accompanied by a lowered lipid clearance from the liver as well as elevated oxidative stress defense and nucleotide degradation. Despite further research still being warranted, our data suggest that GLP-1 may exert insulin-antagonistic effects on hepatic lipid and nucleotide metabolism in ruminants.

  8. Spontaneous Chronic Pain After Experimental Thoracotomy Revealed by Conditioned Place Preference: Morphine Differentiates Tactile Evoked Pain From Spontaneous Pain.

    PubMed

    Hung, Ching-Hsia; Wang, Jeffrey Chi-Fei; Strichartz, Gary R

    2015-09-01

    Chronic pain after surgery limits social activity, interferes with work, and causes emotional suffering. A major component of such pain is reported as resting or spontaneous pain with no apparent external stimulus. Although experimental animal models can simulate the stimulus-evoked chronic pain that occurs after surgery, there have been no studies of spontaneous chronic pain in such models. Here the conditioned place preference (CPP) paradigm was used to reveal resting pain after experimental thoracotomy. Male Sprague Dawley rats received a thoracotomy with 1-hour rib retraction, resulting in evoked tactile hypersensitivity, previously shown to last for at least 9 weeks. Intraperitoneal injections of morphine (2.5 mg/kg) or gabapentin (40 mg/kg) gave equivalent 2- to 3-hour-long relief of tactile hypersensitivity when tested 12 to 14 days postoperatively. In separate experiments, single trial CPP was conducted 1 week before thoracotomy and then 12 days (gabapentin) or 14 days (morphine) after surgery, followed the next day by 1 conditioning session with morphine or gabapentin, both versus saline. The gabapentin-conditioned but not the morphine-conditioned rats showed a significant preference for the analgesia-paired chamber, despite the equivalent effect of the 2 agents in relieving tactile allodynia. These results show that experimental thoracotomy in rats causes spontaneous pain and that some analgesics, such as morphine, that reduce evoked pain do not also relieve resting pain, suggesting that pathophysiological mechanisms differ between these 2 aspects of long-term postoperative pain. Perspective: Spontaneous pain, a hallmark of chronic postoperative pain, is demonstrated here in a rat model of experimental postthoracotomy pain, further validating the use of this model for the development of analgesics to treat such symptoms. Although stimulus-evoked pain was sensitive to systemic morphine, spontaneous pain was not, suggesting different mechanistic

  9. Rat skeletal muscle glycogen degradation pathways reveal differential association of glycogen-related proteins with glycogen granules.

    PubMed

    Xu, Hongyang; Stapleton, David; Murphy, Robyn M

    2015-06-01

    Glycogenin, glycogen-debranching enzyme (GDE) and glycogen phosphorylase (GP) are important enzymes that contribute to glycogen particle metabolism. In Long-Evans Hooded rat whole muscle homogenates prepared from extensor digitorum longus (EDL, fast-twitch) and soleus (SOL, oxidative, predominantly slow twitch), it was necessary to include α-amylase, which releases glucosyl units from glycogen, to detect glycogenin but not GDE or GP. Up to ∼12 % of intramuscular glycogen pool was broken down using either in vitro electrical stimulation or leaving muscle at room temperature >3 h (delayed, post-mortem). Electrical stimulation did not reveal glycogenin unless α-amylase was added, although in post-mortem muscle ∼50 and ∼30 % of glycogenin in EDL and SOL muscles, respectively, was detected compared to the amount detected with α-amylase treatment. Single muscle fibres were dissected from fresh or post-mortem EDL muscles, mechanically skinned to remove surface membrane and the presence of glycogenin, GDE and GP as freely diffusible proteins (i.e. cytoplasmic localization) compared by Western blotting. Diffusibility of glycogenin (∼20 %) and GP (∼60 %) was not different between muscles, although GDE increased from ∼15 % diffusible in fresh muscle to ∼60 % in post-mortem muscle. Under physiologically relevant circumstances, in rat muscle and within detection limits: (1) The total cellular pool of glycogenin is always associated with glycogen granules, (2) GDE is associated with glycogen granules with over half the total pool associated with the outer tiers of glycogen, (3) GP is only ever weakly associated with glycogen granules and (4) addition of α-amylase is necessary in order to detect glycogenin, but not GDE or GP.

  10. Integrating nap and night-time sleep into sleep patterns reveals differential links to health-relevant outcomes.

    PubMed

    Devine, Jaime K; Wolf, Jutta M

    2016-04-01

    Both night-time sleep and nap behaviour have been linked consistently to health outcomes. Although reasons for napping are usually tied to night-time sleep, the majority of studies assess their effects independently. The current study thus aimed to examine the health relevance of patterns of sleep behaviour that take into account both night-time and daytime sleep habits. Night-time sleep, recorded during 7 days via actigraphy from 313 participants (aged 34-82 years) of the Midlife in the United States II Biomarker study, was assessed. Blood and urine specimens were assayed for noradrenaline, interleukin-6 and C-reactive protein. Participants self-reported nap behaviour, depressive symptoms, perceived chronic stress and the presence of medical symptoms and conditions. Overall, nappers (n = 208) showed elevated waist-hip ratios, C-reactive protein and interleukin-6 levels compared to non-nappers and reported more physiological symptoms and conditions (all P ≤ 0.019). Within nappers, cluster analysis revealed three patterns of sleep behaviour-infrequent nappers with good night-time sleep, frequent nappers with good night-time sleep and nappers with poor night-time sleep. Nappers with poor night-time sleep thereby exhibited elevated noradrenaline levels, depressive symptoms and perceived stress scores compared to other groups (all P ≤ 0.041). These findings support the idea that nap-health relationships are complex, in that frequency of napping and accumulation of nap sleep is not related linearly to health consequences. Assessing nap behaviour in conjunction with night-time sleep behaviour appeared crucial to elucidate further the health relevance of napping, particularly in terms of psychological health outcomes, including chronic stress and depressive symptoms.

  11. RNA deep sequencing reveals differential microRNA expression during development of sea urchin and sea star.

    PubMed

    Kadri, Sabah; Hinman, Veronica F; Benos, Panayiotis V

    2011-01-01

    microRNAs (miRNAs) are small (20-23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html.

  12. Maghemite soil nodules reveal the impact of fire on mineralogical and geochemical differentiation at the Earth's surface

    NASA Astrophysics Data System (ADS)

    Löhr, Stefan C.; Murphy, David T.; Nothdurft, Luke D.; Bolhar, Robert; Piazolo, Sandra; Siegel, Coralie

    2017-03-01

    sintered core with low Si, Y/Ho, Zr/Hf and La/Gd and a reciprocal cortex. Preferential loss of the weathering-sensitive cortex, which is rarely preserved on the magnetic nodules, then results in geochemical differentiation of magnetic nodules relative to their non-magnetic precursors. We propose that the elevated Zr/Hf and Y/Ho ratios previously reported for Australian fluvial sediments reflect, at least in part, the long history of palaeo-fires in the catchments of these rivers, with preferential removal, transport and sedimentation of the readily weathered, high Y/Ho and Zr/Hf cortex material that is a product of thermal alteration of Fe nodules. In addition, since magnetic Fe nodules are demonstrably related to fire, they may represent a promising, directly dateable record of severe fires, which can complement the sedimentary charcoal record.

  13. Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes.

    PubMed

    Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun; Yue, David T

    2015-09-01

    Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The sensors are uniformly distributed in neonatal myocytes and respond efficiently to single electrically evoked calcium transients and show cumulative activation at frequencies of 0.5 and 1 Hz. In adult myocytes, the calcineurin sensors appear to be localized to the cardiac z-lines, and respond to cumulative calcium transients at frequencies of 0.5 and 1 Hz. The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes

  14. Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes

    PubMed Central

    Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun; Yue, David T

    2015-01-01

    Abstract The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes. Finally, mathematical modelling combined with quantitative FRET measurements provided novel insights into the kinetics and integration of calcineurin activation in response to myocyte Ca transients. In all, DuoCaN and UniCaN stand as valuable new tools for understanding the role of calcineurin in normal and pathological signalling. Key points Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The

  15. Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5′ splice site selection